Initial Study on the Toxicity of Silver Nanoparticles (NPs) against

Feb 23, 2009 - ... 05 Hradec Kralove, Czech Republic, and Department of Analytical Chemistry, Faculty of Science, Palacky University, Svobody 26, 7714...
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J. Phys. Chem. C 2009, 113, 4296–4300

Initial Study on the Toxicity of Silver Nanoparticles (NPs) against Paramecium caudatum Libor Kvitek,† Marketa Vanickova,† Ales Panacek,† Jana Soukupova,*,† Milan Dittrich,‡ Eva Valentova,‡ Robert Prucek,† Martina Bancirova,† David Milde,§ and Radek Zboril† Department of Physical Chemistry, Faculty of Science, Palacky UniVersity, SVobody 26, 77146 Olomouc, Czech Republic, Department of Pharmaceutical Technology, Faculty of Pharmacy, Charles UniVersity, HeyroVskeho 1203, 500 05 Hradec KraloVe, Czech Republic, and Department of Analytical Chemistry, Faculty of Science, Palacky UniVersity, SVobody 26, 77146 Olomouc, Czech Republic ReceiVed: September 30, 2008; ReVised Manuscript ReceiVed: January 5, 2009

In this initial study, the toxicity effect of silver NPs against a model unicellular eukaryotic organism of Paramecium caudatum was studied. For the purpose of this study, a dialysis-based method was adapted, which allowed the preparation of stable aqueous dispersions of silver NPs in various silver concentrations that were necessary for the evaluation of toxicity limits of these particles. The obtained results demonstrate that the silver NPs do not exhibit any toxicity action against the tested unicellular eukaryotic organism below the concentration of 25 mg · L-1 whereas ionic silver retains its toxicity even at a concentration of 0.4 mg · L-1. Such a considerable difference in the toxicity effect of these two forms of silver has not been observed in the previously published study concerning bacteria (Pana´cˇek, A.; Kvítek, L.; Prucek, R.; Kola´ˇr, M.; Vecˇerˇova´, R.; Pizu´rova´, N.; Sharma, V. K.; Neveˇcˇna´, T.; Zborˇil, R. J. Phys. Chem. B 2006, 110, 16248-16253). Additionally, it was proven that the surfactant/polymer modification can increase the toxicity of the silver NPs against the tested organism. Introduction Since the beginning of the 21st century, huge progress in the synthesis and application of new nanomaterials based on carbon, metals, and their oxides has been made. These materials have quickly expanded into our everyday life; therefore, a question considering both human and environmental toxicity has consequentially emerged. Possible toxicological risks of these materials are undoubtedly reflected in the establishment of a new scientific fieldsnanotoxicology.1-3 Research on the environmental toxicity of nanotechnological products, in spite of their rapid development, is currently taking its first hesitant steps.4,5 So far, only a few articles have addressed the problem of possible environmental toxicity of silver NPs.6,7 Thus, although silver NPs have been widely used as antibacterial agents in fabric coatings and as surface modifiers of other devices in everyday use,8,9 their environmental toxicity remains an unsolved question. The toxicity effect of silver NPs has already been tested on bacteria10 on one side and on mammalian cells on the other side;11,12 however, there still exist a variety of simple or more complex organisms that are very important to the establishment of a balance in the environment and against which the toxicity of silver NPs has not yet been investigated. For example, the unicellular eukaryotic organism of Paramecium sp. represents one of the significant indicators of water pollution caused by heavy metals.13-16 On the basis of the above-mentioned reasons, this initial study evaluating the environmental toxicity of the silver NPs employed Paramecium caudatum as a model organism. The aqueous dispersion of silver NPs was prepared via a modified Tollens process.17 The as-prepared dispersion was concentrated using * Corresponding author. E-mail: [email protected]. Tel: +420 585 634 420. Fax: +420 585 634 425. † Department of Physical Chemistry. ‡ Department of Pharmaceutical Technology. § Department of Analytical Chemistry.

an innovatively adapted dialysis-based procedure. The toxicity impact of the silver NPs and ionic silver (as a reference sample) is expressed as the dependence of the lethality time of 50% of the tested organisms (LT50) on silver concentration. The obtained results were compared with the previously published study on the antibacterial effect of silver NPs.10 Finally, we evaluated the possible influence of surface modification, performed with chosen surfactant/polymers, on the toxicity of the silver NPs against the tested organism of P. caudatum. Experimental Section Materials. The silver NPs were prepared via a modified Tollens process using the following chemicals: silver nitrate (99.9%, Safina), ammonia (p.a., 25% w/w aqueous solution, Lachema), sodium hydroxide (p.a., Lachema), and D(+)-maltose monohydrate (p.a., Riedel-de Hae¨n). For the purpose of the modification of the silver NPs, the following substances were used: nonionic surfactant polyoxyethylene (20) sorbitan monooleate (Tween 80; p.a., Lachema), polyethylene glycol with a molecular weight of 35 000 (PEG 35000) (p.a. Fluka), and polyvinylpyrrolidone having a molecular weight of 360 000 (PVP 360) (p.a., Sigma-Aldrich). In the concentrating procedure, a superabsorbing poly(acrylate-co-vinylalcohol) copolymer (p.a., Sigma-Aldrich) was wrapped in the dialysis tubing cellulose membranes (average flat width of 25 mm, Aldrich). The abovementioned chemicals were used without any further purification. All of the used solutions were prepared with deionized water (18 MΩ · cm, Millipore). The pure laboratory culture of the unicellular eukaryotic organism of P. caudatum was supplied by the Faculty of Medicine, Masaryk University in Brno (Czech Republic). Instrumentation. The size and zeta potential of the silver NPs were measured by the method of dynamic light scattering (Zetasizer Nano ZS, Malvern). The size of the silver NPs was also confirmed by TEM (JEM 2010, Jeol) and by UV/vis

10.1021/jp808645e CCC: $40.75  2009 American Chemical Society Published on Web 02/23/2009

Ag Nanoparticle Toxicity against Paramecium caudatum

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Figure 1. Scheme of the concentrating procedure of the aqueous silver NP dispersion using the superabsorbing copolymer wrapped in dialysis membranes.

absorption spectra (Specol S 600, Analytic Jena). Atomic absorption spectrometry (spectrometer Avanta Σ (GBC)), applying flame atomization with an acetylene-air flame, was used for the evaluation of the content of silver in the aqueous dispersions. Synthesis of Silver Nanoparticles. The aqueous dispersion of the silver NPs with a concentration of 108 mg · L-1 was prepared by the reduction of the complex cation [Ag(NH3)2]+ with D-maltose in alkali media (pH 11.5).17 The reduction was performed at laboratory temperature (∼25 °C) with vigorous mixing. The initial concentrations of the reaction components were 1 × 10-3 and 5 × 10-3 mol · L-1 for AgNO3 and NH3, respectively. The concentrations of NaOH and maltose were 1 × 10-2 mol · L-1. The synthesis of the silver NPs was completed within 5 min. The concentration of free Ag+ in the final silver NP dispersion was determined by potentiometric measurements using a silver ISE electrode (Radiometer Analytical SAS, France) as the indicator electrode and a saturated calomel electrode with a saturated KNO3 salt bridge as the reference electrode. The concentration of free Ag+ did not exceed the value of 1 × 10-7 mol · L-1, which is the detection limit of the ISE electrode used. Concentrating Procedure. For the purpose of the toxicity study, a dialysis-based method was adapted that enabled us to obtain more concentrated dispersions of silver NPs. In the course of this innovatively used process, dialysis tubing cellulose membranes (average flat width of 25 mm) filled with superabsorbing poly(acrylate-co-vinylalcohol) copolymer were applied. The concentrating procedure was initiated with seven dialysis tubing cellulose membranes, each filled with 1 g of the superabsorbing copolymer and inserted into 500 mL of the asprepared silver NP dispersion (Figure 1). The as-prepared dispersion of silver NPs had a starting silver concentration of 109 mg · L-1 (as determined by AAS; the calculated concentration was 108 mg · L-1). After 36 h of the concentrating procedure, new dialysis tubing cellulose membranes replaced the initially used ones, which contained the swollen superabsorbing copolymer. The concentrating procedure was monitored throughout samples taken at regular time intervals (Table 1). At these intervals, the samples were characterized by the following characteristics: pH, conductivity, size, size distribution, zeta potential, and UV/vis absorption spectra. The procedure was stopped after 84 h, reaching a silver concentration of 718 mg · L-1 as determined by AAS. Note that for the purpose of the monitoring measurements it was necessary to dilute the concentrated samples in order to

TABLE 1: Fundamental Physico-Chemical Characteristics of the Aqueous Silver NP Dispersions in the Course of the Concentrating Procedure Performed via the Dialysis-Based Method Using Tubing Cellulose Membranes Stuffed with Superabsorbing Copolymer concentration time of of silver in particle size/ zeta concentration the dispersion half-width potential conductivity (h) (mg · L-1)a (nm)b (mV) (mS · cm-1) pH 0 4 8 12 24 36 48 60 72 84

109.1 116.3 121.4 125.2 169.5 235.7 288.3 375.3 472.1 718.1

27/8 31/13 31/12 32/13 32/12 31/11 36/16 35/15 39/19 40/21

-37 -40 -38 -37 -38 -39 -38 -36 -34 -42

1.23 0.90 0.80 0.74 0.65 0.58 0.52 0.44 0.42 0.33

11.5 10.7 10.1 9.7 8.8 6.9 6.8 6.5 6.5 6.3

a Concentration of total silver in the concentrated dispersion sample was determined by AAS. b Average diameter of the silver NPs in the aqueous dispersion was determined by DLS as the z average, and this value is completed by the value of the half-width of the log-normal distribution peak fitting the data obtained from DLS.

avoid measurement inaccuracy (UV/vis spectroscopy) or even to enable measurements of the size, size distribution, and zeta potential using the DLS method. In the case of measurements using the DLS method, samples of the concentrated dispersions would be immeasurable. Therefore, all of the samples taken in the course of the concentrating procedure were diluted to the initial concentration of 109 mg · L-1. In the case of UV/vis measurements, dilution to 109 mg · L-1 would not produce reliable data because of the extreme absorbance; therefore, these samples were additionally diluted to a concentration of 10 mg · L-1. The above-mentioned dilution was conducted for characterization purposes only. The toxicity tests were carried out with a concentrated dispersion of silver NPs. Toxicity Assays. The toxicity assays employed aqueous dispersions of the silver NPs/ionic silver/modified silver NPs that were tested against the unicellular eukaryotic organism P. caudatum. The toxicity was determined as the lethality time of 50% organisms (LT50). LT50 was measured from the moment of the addition of the particular silver form into the culture of P. caudatum up to the moment when 50% of the organisms died. In a typical toxicity assays, 1-5 mL of the P. caudatum

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Figure 2. Representative TEM images of (a) an as-prepared silver NPs dispersion and (b) after 36 h of the concentrating procedure.

culture (200-300 organisms/mL) was inserted into a test tube. Afterward, 0.05-1 mL of the particular silver form, in various concentrations, was added, and the test tube was vigorously shaken by hand. Such a mixture (200 µL) was immediately transferred onto the microscopic glass slide. A set of approximately 50 organisms were monitored over an area of 1 × 1 cm using an optical microscope at low magnification (40×). All of the experiments evaluating the toxicity of the silver NPs were repeated three times, and the results are expressed as the average value of the three observations. The standard deviation of the obtained LT50 value did not exceed 1%. Additionally, the toxicity of the aqueous dispersions of the silver NPs was evaluated as the concentration leading to 50% lethality of the tested organisms assessed after 1 h of contact with the silver NP dispersion (1 h LC50). This value was obtained from the dependence of LT50 on the concentration of silver (in the form of NPs) in the dispersion. Results and Discussion Concentration Procedure. Preliminary tests evaluating the toxicity of the silver NPs (with the silver concentration in the aqueous dispersion equal to 108 mg · L-1) against the tested P. caudatum revealed a significantly lower effect against this eukaryotic organism than against the previously tested bacteria.10 Therefore, the consequential toxicity assays had to be preceded by the development of a suitable concentrating method, which could ensure that more concentrated silver NP dispersions were obtained. The dialysis-based method, originally used for the concentration of aqueous dispersions of biomacromolecules, was finally found to be applicable to an inorganic NP dispersion. This method uses a superabsorbing poly(acrylate-co-vinylalcohol) copolymer wrapped in a dialysis tubing cellulose membrane and enables us to obtain more concentrated silver NP dispersions with negligible changes in the particle characteristics. The experiments that were performed proved that the aggregation stability of the silver NPs is strongly dependent on the rate of the concentrating process, which is directly related to the amount of superabsorbing copolymer used. The optimized amount of 7 g of superabsorbing copolymer per 500 mL of NP dispersion after 36 h was adjusted to ensure the preservation of the fundamental characteristics (average size, polydispersity, and zeta potential) of the as-prepared silver NP dispersion. When a larger amount of superabsorbing copolymer was used, the increased rate of the concentrating process resulted in a rapid aggregation process. The aggregation stability of the silver NPs is clearly demonstrated by TEM images (Figure 2). The observed spherical particles reveal the comparable average size (∼30 nm), both in the as-prepared and 36 h concentrated dispersions. Similarly, the DLS data (Table 1) reflect the precise

Figure 3. (A) UV/vis absorption spectra of the as-prepared silver NP dispersion and corresponding spectra after (B) 24, (C) 48, and (D) 72 h of the concentrating procedure.

preservation of the average particle size up to 36 h, which can be considered to be negligible. Thus, we obtained a sufficiently large concentration range of silver NPs with nearly the same size characteristics. This enabled the evaluation of their toxicity limits against the higher organism P. caudatum. The preservation of the average size of the variously concentrated silver NP systems was also checked through minor changes in the position of the surface resonance plasmon peak in the recorded UV/vis spectra (Figure 3). Its location at approximately 400 nm corresponds to NPs with diameters of 30-40 nm.18 This extraordinary aggregation stability of the concentrated silver NP dispersions is undoubtedly connected with the removal of dissolved electrolytes from the dispersion. The removal of excess electrolytes reduces the ionic strength that is responsible for the aggregation of the silver NPs as a result of the suppression of interparticle repulsion. It is in accordance with the theory of colloid stability formulated by Derjaguin, Landau, Verwey, and Overbeek (DLVO theory).19 The gradual removal of the present electrolytes from the dispersion is well documented by a steep decrease in conductivity and also by a significant change in the pH of the samples from an initial value of 11.5 to 6.9 (optimal for the tested organism) gained after 36 h (Table 1). In comparison, the same change in pH induced by the neutralization of the as-prepared silver NP dispersion led to the formation of larger agglomerates and to an increase in the polydispersity of the system. When pH was adjusted in this way to 6, the particle size increased by

Ag Nanoparticle Toxicity against Paramecium caudatum

Figure 4. Dependence of LT50 of P. caudatum on the concentrations of unmodified silver NPs (O A), ionic silver (• B), and Tween 80 modified silver NPs (0 C) in the tested systems.

a factor of 2 because of the induced aggregation of silver NPs into larger aggregates. This is a further proof that the suitably adjusted dialysis-based concentration method represents a unique, powerful approach to the preparation of several times more concentrated silver NP dispersions that retain almost the same size characteristics. These dispersions are stable for several months. Toxicity Assays. The second part of this study was devoted to the evaluation of the toxicity of the unmodified and modified silver NPs against unicellular organism P. caudatum. Before discussing the toxicity of silver NPs against P. caudatum, it is necessary to characterize the experimental parameters used for the quantification of the toxicity effect in this study. The 24 h LC50 value, as the most commonly used quantity in toxicological studies, is not exactly suitable in this case because of the very steep change in resistance of the tested unicellular organism against the silver NPs when their concentration falls to the limit of the toxic action. For that reason, we used the 1 h LC50 parameter to quantify the toxicity effect of the silver NPs against P. caudatum more precisely. This parameter was simply determined from the dependence of the LT50 on concentration. The results obtained primarily on the unmodified silver NPs have shown a large decrease in toxicity against the tested organisms at concentrations of silver NPs below 200 mg · L-1, which was reflected in the prolonged LT50 (Figure 4). Even at a concentration of 25 mg · L-1, the organisms were able to survive for more than 7 days; therefore, this value can be considered to be the lower boundary of the toxicity effect of the silver NPs against the tested organism. Finally, the 1 h LC50 value, representing the key parameter for the quantification of the environmental toxicity of the silver NPs, was found to be 39 mg · L-1. Furthermore, the obtained results can be compared with the previously published study of the antibacterial activity of the silver NPs. The values of the minimal bactericidal concentrations of silver (in the form of NPs) ranged from 1.69 to 13.5 mg · L-1 depending on the tested bacterial strain; the lowest value was achieved with Staphylococcus epidermidis, and the highest, with Enterococcus sp.10 These values of the minimal bactericidal concentrations can be compared with the hereby determined 1 h LC50 value (39 mg · L-1) because the silver NPs were prepared

J. Phys. Chem. C, Vol. 113, No. 11, 2009 4299 via the same, well-established method and therefore had comparable particle characteristics. In this way, it was proven that the toxicity of the silver NPs is obviously higher against the prokaryotic organisms (i.e., bacteria) than against the tested unicellular eukaryotic organism. Second, we tried to compare the toxicity of silver NPs and ionic silver against P. caudatum. The solution of AgNO3 was used as a reference sample representing the most toxic form of silver. A concentrations of ionic silver as low as 0.4 mg · L-1 caused the immediate death of the P. caudatum organism (Figure 4B). In contrast to these results, even more than a 50-fold higher concentration of silver NPs (25 mg · L-1) did not reveal any acute toxicity effect against the tested unicellular eukaryotic organism. This selective toxicity effect of different forms of silver was not observed to such an extent in the previously published study on antibacterial activity.10 Therefore, this newly gained knowledge of the selective action of silver NPs, in contrast to the nonselective action of ionic silver, is one of the key conclusions of this initial study, especially for the forthcoming applications based on the antibacterial properties of silver NPs or silver compounds. Because the silver NPs can be modified by surfactants/ polymers (purposely in order to enhance their stability or incidentally in the environment), the last part of this study was necessarily devoted to the investigation of possible changes in the toxicity of the surfactant/polymer-modified silver NPs. For this purpose, we investigated the following modifiers: one of the commonly used a nonionic surfactant (Tween 80) and also two polymers (PEG 35000 and PVP 360). Primarily performed toxicity tests with a reference sample of “pure” 1% (w/w) solutions of the modifiers that were used (without silver NPs) showed no toxicity effect against P. caudatum. However, if the silver NPs were modified by Tween 80 (1% w/w), then this enhanced the toxicity of these silver NPs against the tested unicellular organism. This synergic effect can be demonstrated throughout the change in LT50 for the silver NPs modified by Tween 80 (Figure 4C). This enhancement of the toxicity effect of the Tween 80-modified silver NPs can be quantified via a decrease in the 1 h LC50 from 39 mg · L-1 for the unmodified silver NPs to 16 mg · L-1 for silver NPs modified by Tween 80. On the contrary, the modification of silver NPs by PEG 35 000 led to only a slight change in the toxicity against P. caudatum compared to that of the unmodified system (part A vs part B of Figure 5). Nevertheless, in the case of the silver NPs modified by PVP 360, a certain increase in toxicity with silver concentrations between 50 and 200 mg · L-1 (Figure 5C) was observed. However, the highest and lowest tested concentrations of the aqueous dispersions of the silver NPs modified by PEG and PVP had an acute toxicity effect comparable to that of the unmodified silver NPs. Therefore, the values of the 1 h LC50 remained comparable with the system containing unmodified silver NPs. The above-mentioned slight differences in toxicity of the PEG- and PVP-modified silver NPs can be explained by different interaction modes of the polymers with the surface of the NP. Whereas PEG interacts with the silver surface through the oxygen atom in its molecule, PVP interacts in a stronger way through the nitrogen atom.20 The stabilizing effect of these polymers is determined by the strength of these interactions, which has already been confirmed by a study aimed at finding the relationship between the stabilizing effects of polymers/surfactants on silver NPs and their antibacterial activity.20 The insignificant effect of polymer modification on the toxicity of silver NPs against the tested organism is in sharp

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Kvitek et al. against the prokaryotic bacteria. Beside these facts, it has been proven that the surface modification of the silver NPs was responsible for the increase in toxicity against the tested unicellular eukaryotic organism, as demonstrated especially by nonionic surfactant Tween 80. Thus, the environmental toxicity risks related to the use of silver NPs in everyday life should not be disdained. Additionally, appropriate attention should be paid to the possible impact of the interaction of surfactants from wastewater with silver NPs. This interaction can lead not only to the enhancement of their stability but mainly to an increase in their toxicity effect. Acknowledgment. Financial support from the Ministry of Education of the Czech Republic (MSM6198959218, MSM0021620822, 1M6198959201, and MSM6198959216) is gratefully acknowledged. References and Notes

Figure 5. LT50 of unicellular eukaryotic organism P. caudatum exposed to different concentrations of unmodified (O A), PEG 35 000- (• B), and PVP 360-modified (0 C) silver NPs.

contrast to the observed synergic effect of nonionic surfactant Tween 80. Such a difference in the behavior of the surfactant/ polymer-modified silver NPs can be ascribed to a different mode of interaction with the tested unicellular eukaryotic organism. Whereas the surfactant is suspected from the easier penetration of the cell wall of the tested unicellular eukaryotic organism, the polymers are supposed to prevent only the aggregation process of the silver NPs. Therefore, the polymers can retain the biological activity of the silver NPs, which is strongly dependent on the particle size.21 Conclusions We have introduced a suitable dialysis-based concentrating procedure enabling the preparation of highly concentrated aqueous dispersions of silver NPs while retaining the fundamental particle characteristics. The toxicity of the silver NPs against unicellular eukaryotic organism P. caudatum is significantly lower than the toxicity against previously tested bacterial strains and can be quantified by a 1 h LC50 of 39 mg · L-1. The silver NP dispersion, prepared via the same reduction process, effectively kills common bacterial strains in concentrations of silver that are approximately 3-10 times lower. Moreover, the tested silver NPs were revealed to act selectively. Whereas ionic silver proved to be toxic to both bacterial strains and the tested unicellular organism, silver NPs revealed high toxicity only

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