doi:10.1111/iej.12377
Positively charged imidazolium-based ionic liquid-protected silver nanoparticles: a promising disinfectant in root canal treatment
A. Abbaszadegan1, M. Nabavizadeh1, A. Gholami2, Z. S. Aleyasin3, S. Dorostkar4, M. Saliminasab5, Y. Ghasemi2, B. Hemmateenejad4 & H. Sharghi4 1
Department of Endodontics, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz; 2Department of Pharmaceutical Biotechnology and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz; 3Department of Oral Medicine, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz; 4Department of Chemistry, Shiraz University, Shiraz; and 5Shiraz Dental School, Shiraz, Iran
Abstract Abbaszadegan A, Nabavizadeh M, Gholami A, Aleyasin ZS, Dorostkar S, Saliminasab M, Ghasemi Y, Hemmateenejad B, Sharghi H. Positively charged imidazolium-based ionic liquid-protected silver nanoparticles: a promising disinfectant in root canal treatment. International Endodontic Journal.
Aim To synthesize and characterize silver nanoparticles (Ag NPs) with different surface charges in order to evaluate their cytotoxicity and antibacterial activity in the absence and presence of dentine compared with NaOCl and CHX. Methodology Ag NPs with positive, negative and neutral surface charges were synthesized and characterized. The first phase of the experiment determined the minimum inhibitory concentrations (MICs) of NPs against planktonic E. faecalis and compared them with that of NaOCl and CHX. The second phase tested the elimination of E. faecalis at different contact times (5, 20 and 60 min and 4 and 24 h), and the role of dentine in their inactivation was assessed. In the third phase, the most effective Ag NP solution was selected for cytocompatibility assessment. An MTT-based cytotoxicity assay was used to evaluate the cytotoxicity of the selected NP solution in different concentrations on L929 fibroblasts compared to that of
2.5% NaOCl and 0.2% CHX. Student’s t-test and repeated measures MANOVA approach were used for statistical analyses. Results The characterization revealed synthesis of colloidal NPs in the size range of 5–10 nm in diameter. The results indicated that Ag NP with a positive surface charge had the smallest MIC against planktonic E. faecalis, and it was active in very lower concentrations compared to NaOCl, CHX and the other tested AgNPs. Positive-charged Ag NPs at 5.7 9 10 10 mol L 1 completely prevented the growth of E. faecalis after 5 min of contact time, a finding comparable to 0.025% NaOCl. Dentine powder had variable inhibitory effects on all tested materials after 1 h incubation period, but after 24 h, NaOCl and the positive-charged Ag NPs were not inhibited by dentine at any concentration used. CHX was the most and the positively charged Ag NP solution was the least toxic solutions to L929 fibroblasts (P < 0.001). Conclusions Ag NP surface charge was important in bactericidal efficacy against E. faecalis. The positively charged imidazolium-based ionic liquidprotected Ag NPs showed promising antibacterial results against E. faecalis and exhibited a high level of cytocompatibility to L929 cells. Keywords: antimicrobial, cytotoxicity, dentin, Enterococcus faecalis, ionic liquid, silver nanoparticles. Received 24 February 2014; accepted 27 August 2014
Correspondence: Mohammadreza Nabavizadeh, Ghomabad St, Ghasrodasht St, Dental School, Shiraz, Iran (Tel.: +989173029846; Fax: +987116263192; e-mail: nabaviza [email protected]).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
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Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
Introduction Nanotechnology continues to have a critical impact in all disciplines including dental sciences. Consequently, endodontics can benefit from this field of research due to the various chemical and physical properties of the materials produced on a nanoscale. Recent studies have reported the strong antibacterial activity of nanoparticles such as chitosan and zinc oxide against Enterococcus faecalis (Kishen et al. 2008, Shrestha et al. 2010). Silver nanoparticles (Ag NPs) have also drawn the attention of researchers in recent endodontic investigations. The antibacterial activity of Ag NPs against several species of endodontic bacteria has been recorded in several investigations (Kim et al. 2007, Shrivastava et al. 2007, Lotfi et al. 2013, Wu et al. 2014). Besides, antibacterial nanoparticles do not seem to provide the bacteria with the capacity to gain resistance (Rai et al. 2012). Therefore, they may serve as promising disinfectants for endodontic purposes. Nanoparticles (NPs) can be synthesized by various methods which influence the particle’s characteristics including morphology, surface charge, stability and chemical reactivity (Chaloupka et al. 2010). The antimicrobial properties of several metallic NPs are well documented. This feature may be influenced by variables including the size, shape and the surface charge of the particles (Tran & Le 2013). Also, the same variables play a substantial role in nanotoxicity (Tran & Le 2013). There is no comprehensive study in the literature to assess the cytotoxicity and antimicrobial activity of Ag NPs with different surface charges against E. faecalis relative to other endodontic irrigants to enlighten the position of these particles in future endodontic practice. Likewise, reports focusing on the surface charge of Ag NPs and its relevance to antimicrobial effectiveness have proven inconclusive. In this study, stable Ag NPs with different sizes and surface charges were synthesized and characterized. The antibacterial activity and cytotoxicity of these products were compared with sodium hypochlorite (NaOCl) and chlorhexidine (CHX), and the role of dentine in inactivation of these irrigants was assessed. The null hypotheses tested were as follows: (i) there is no difference in antibacterial efficacy of differently charged Ag NPs against planktonic E. faecalis when compared with NaOCl and CHX, (ii) dentine has no inhibitory effect on antibacterial activity of these irrigants, and (iii) there is no difference amongst the
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cytocompatibilities of these irrigants against L929 mouse fibroblasts.
Materials and methods Synthesis of neutral silver NPs The neutral Ag NPs (Neut Ag NPs) were synthesized using the method described by Zandi-Atashbar et al. (2011) in which the milled rice samples were used directly with no sample pre-processing. Powdered rice samples were dried by storing at 60 °C overnight. A 100.0 mg portion of the rice powders was added into a test tube containing 10.0 mL buffer solution whilst the tubes were placed in a boiling water bath for 15 min. Furthermore, 1.0 mL of AgNO3 (0.01 molar) was added, and the resulting suspension was heated for an additional 5 min, until the colour of the solution became golden.
Synthesis of negative-charged silver NP Negative-charged Ag NPs (NC Ag NPs) were synthesized through the reduction of silver nitrate using sodium borohydride. All glassware was placed in 1 : 3 HCl/HNO3 solution and then rinsed with triple distilled water for three times. Furthermore, the sodium borohydride was chilled to roughly 0 °C using ice and stirring. A 1.0 mL portion of 0.01 m mol L 1 AgNO3 was added to 20 mL of the stirring 6.2 m mol L 1 NaBH4 solution using a dropper, at approximately one drop per second. Stirring was stopped after complete addition of AgNO3.
Synthesis of positive-charged silver NPs The ionic liquid-protected silver nanoparticles (PC Ag NPs) were prepared according to the procedure described by Hemmateenejad et al. (2013). In brief; all glassware was placed in 1 : 3 HCl/HNO3 solution, rinsed with triple distilled water three times; then, 1.0 mL of 0.01 mol L 1 AgNO3 aqueous solution was added to 20 mL of 6.2 m mol L 1 1-dodecyl-3-methylimidazolium chloride ([C12mim][Cl]) aqueous solution, and the solution was stirred vigorously. The freshly prepared 0.4 mol L 1 NaBH4 aqueous solution was then added to the stirred solutions dropwise until the colour of the solution became golden. Subsequently, the colloidal solutions were centrifuged for 20 min to remove excess amount of ionic liquids. The resultant goldencoloured solution was stored at room temperature.
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Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
Characterization of Ag NPs As the NP characterization is necessary to establish the understanding and control of NP synthesis and application, the formation of Ag NPs was evidenced by UV–visible spectra of the colloidal silver solutions. Absorption spectra were recorded on an Ultrospec 3000 UV–vis spectrophotometer (Pharmacia Biotech, San Francisco, CA, USA) equipped with a 1 cm quartz cell. Furthermore, to have a better estimation of the size range of the prepared Ag NPs, they were characterized with transmission electron microscopy. The measurements were made on a transmission electron microscope (TEM, JEM 2010, Jeol, Tokyo, Japan) operated at an accelerating voltage of 100 kV. Samples for TEM characterization were prepared by placing a drop of the as-prepared silver colloidal samples (wellcentrifuged samples) on a carbon-coated copper grid and then drying at room temperature. Several TEM images were prepared for each synthesized Ag NP, and the average size of 250 particles in these images was recorded. Using the size and absorbance of the NPs, the concentrations of the aqueous solution of the synthesized NPs were calculated according to the procedure suggested by Zhang et al. (2009a). The surface charge or zeta potential of the synthesized NPs was verified by zeta potential analysis using zeta potential analyser (Zeta Plus, Brookhaven Instrument Co., Holtsville, NY, USA).
Measurement of antibacterial activity against E. faecalis Medicaments The experimental groups were consisted of negative, neutral and positive-charged Ag NPs at their initial synthesized concentrations, 2.5% NaOCl (SigmaAldrich Co., St. Louis, MO, USA) and 0.2% CHX (Sigma-Aldrich Co.). All medicaments were then diluted from their initial concentrations. Sterile water was used as negative control. All medicaments were freshly prepared prior to commencement of experiments. Test organism The test organism in this study was E. faecalis strain AGH 011, isolated from a previously root filled tooth with persistent apical periodontitis (GeneBank: KF701470). The strain was identified by gram
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
staining, catalase reaction, colony morphology and by the patterns of carbohydrate fermentation and enzyme production. An overnight culture on TSA plates (Tryptic-soy-agar, Difco, MI, USA) was checked for purity, suspended in 0.5% peptone water (Bacto peptone, Difco) and adjusted spectrophotometrically to match the turbidity of 2 9 109 CFU mL 1. Minimum inhibitory concentration (MIC) determination MIC determination of each medicament against E. faecalis was conducted three times (triplicate) by a microdilution method with Mueller-Hinton broth (Himedia, Mumbai, India) supplemented with calcium (25 mg L 1) and magnesium (12.5 mg L 1). All procedures were performed according to the guidelines presented by the Clinical and Laboratory Standards Institute (CLSI) (Wikler 2010). 10-fold serial dilutions of each medicament were prepared (up to eight times). Then, the MIC50 and MIC90 were defined as the lowest concentration, which inhibited 50% and 90% of the growth when compared with growth control, respectively. Elimination of E. faecalis in different contact times For the first phase of the experiment, 950 lL of each experimental solution was mixed with 50 lL of bacterial suspension in an Eppendorf test tube. In this step, imidazole solution was also employed as an experimental solution because of its role in PC Ag NPs synthesis. These suspensions were carefully mixed and incubated at 37 °C in air. Furthermore, 10 lL of each sample was collected after determined incubation periods (5, 20 and 60 min and 4 and 24 h). Each sample was utilized for preparing 10-fold serial dilutions (up to five times). Each dilution was cultured on a TSA plate at 37 °C for 24 h, and following, the plates were checked for the bacterial growth under a stereomicroscope. The number of colonies was calculated, and the bacterial growth was measured by the CFU mL 1 counts of E. faecalis. Microscopic observation of the colonies was carried out to rule out any contamination. These procedures were repeated for three times to reduce the possible bias.
Evaluation of the inhibitory effect of dentine powder on the antibacterial activity of the medicaments This protocol was adopted from Portenier et al. (2006). Freshly extracted human third molar teeth were collected and kept in 0.5% NaOCl for 1 h to
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clear soft tissue residues and to stop microbial growth. The teeth were rinsed with distilled water and then autoclaved at 121 °C for 15 min. The roots were separated from the crowns and sectioned to produce small slices using a low-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA). The root slices were then grounded by a planetary ball-milling device (Retschâ PM400, Retsch GmbH, Haan, Germany) at 400 rpm for 6 h to yield the dentine powder with a particle size of 0.2–20 lm in diameter. Selected concentrations of the medicaments were employed to include in this part of study based on data obtained from their effectiveness in the absence of dentine. In an Eppendorf tube, per experiment, a total of 28 mg of dentine powder was suspended in 50 lL sterile water, then, carefully mixed with 50 lL of the bacterial suspension (1.0 9 108 CFU) together with 50 lL of each medicaments (total volume: 150 lL). In control tubes, dentine powder was not used and another 50 lL of sterile water was added to keep the total volume of 150 lL. The suspensions were incubated at 37 °C in an aerophilic environment for 1 h and 24 h. Furthermore, 10 lL of each sample was cultured, and the number of colonies was counted. This procedure was performed in triplicate.
sulphoxide (DMSO) was added to each well. The absorption of the solution was at a wavelength of 540 nm by an ELISA plate reader (PowerWaveTM X52, BioTek Instruments Inc., Potton, UK). This indirect cytotoxicity test was performed three times for each sample. The mean optical density values (OD) of three wells containing the same extract with their standard deviations were calculated. The mean cell viability values were defined as the percentage of the OD values of the negative control.
Statistical analysis The data obtained from the effectiveness of the experimental solutions, in the absence and presence of dentine, were evaluated for the statistical significance using the Student’s t-test. Repeated measures MANOVA approach and subgroup analysis were used to evaluate the differences in the mean cell viability values of L929 fibroblasts. For the purpose of subgroup analysis, one-way ANOVA/Tukey tests were used. A level of P < 0.05 was accepted as statistically significant.
Results Characterization of Ag NPs
Cytotoxicity of the medicaments on L929 fibroblasts Cytotoxicity of the medicaments on L929 mouse fibroblasts was evaluated using MTT colorimetric assay. Three different concentrations of PC Ag NPs ranging from 5.7 9 10 8 to 5.7 9 10 10 mol L 1, 2.5% NaOCl and 0.2% CHX were selected. Culture medium and 35% hydrogen peroxide were regarded as the negative and positive controls, respectively. L929 mouse fibroblasts in RPMI 1640 media were aliquoted into a 96-well cell culture plate at a density of approximately 1 9 104 cells/well and incubated in a humidified atmosphere of 5% CO2, 95% air at 37 °C to reach roughly 80% confluence. After 24 h, 100 lL of each sample which was previously incubated at 37 °C in RPMI 1640 media for 24 h was transferred into each well. The medium was removed at 1, 4, and 24 h after incubation, and the wells were washed twice for 2–3 min with 200 lL of the fresh medium. A total of 25 lL of MTT [3-(4,5 Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] (Sigma-Aldrich Co.) stock solution was added to each well and incubated in a humidified atmosphere of 5% CO2, 95% air for 4 h at 37 °C. Then, 100 lL of dimethyl
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The peak of the plasmon resonance band of the synthesized Ag NPs was located at about 400 nm (Table 1) attributing to the formation of Ag NPs in the size ranges of 5–10 nm in diameter (Zhang et al. 2009a). The sample TEM images are given in Fig. 1, and the average sizes of the synthesized Ag NPs, based on the TEM images, and their initial concentrations are listed in Table 1. The mean particle sizes measured based on particle counting were in good agreement with those estimated by absorbance peak of the surface plasmon resonance bands. However, for the Neut Ag NPs, an overlap between absorbance of starch and surface plasmon resonance of Ag NPs made the estimation of the net absorbance contribution of Ag NPs impractical. Consequently, determination of the exact concentration of Ag NPs was unfeasible. The zeta Table 1 Properties of the synthesized Ag NPs Surface charge
kmax (nm)
Absorbancemax (nm)
Average size (nm)
Negative Neutral Positive
400 425 407
0.45 ND 1.8
7.5 10.1 9.0
Concentration (mol L 1) 9.7 9 10 4.0 9 10 5.7 9 10
8 8 8
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
(a)
(b)
(c)
Figure 1 Sample TEM images of negative (a), positive (b) and neutral (c) Ag NPs.
potential of the NC Ag NPs was obtained as -38.0 mV, whereas the surface charges of the synthesized PC Ag NPs and Neut Ag NPs were determined as +50.0 mV and 0.0 mV, respectively. These values of zeta potential represent a good stability for the charged NPs (Greenwood & Kendall 1999).
Minimum inhibitory concentration determination Table 2 demonstrates the MIC determination for E. faecalis when exposed to each tested medicament.
Elimination of E. faecalis at different contact times Enterococcus faecalis had 100% growth when treated with sterile water as control. For the experimental groups, the survival rate of E. faecalis was expressed as a percentage of survival in the control group. Different concentrations of imidazole showed 100% growth for E. faecalis in all defined incubation periods. The antibacterial effect of other solutions was dependent on their concentration and contact time. 0.2% CHX completely inhibited the growth of E. faecalis at all incubation periods. PC Ag NPs at 5.7 9 10 10 mol L 1 and NaOCl at 0.025% completely prevented the growth of E. faecalis even after 5 min of contact time. NC Ag NPs at full strength concentration (9.7 9 10 8 mol L 1) started to inhibit the growth of E. faecalis after 1 h of contact time. Neut Ag NPs had moderate inhibitory effect on E. fae-
Table 2 The minimum inhibitory concentrations (mol L
1
)
of the tested medicaments against E. faecalis Medicaments
MIC
CHX NaOCl PC Ag NPs NC Ag NPs Neut Ag NPs
9 9 9 9 9
2.4 3.7 5.7 9.7 4.0
MIC
50
10 10 10 10 10
5 7 15 8 9
calis dependent on their concentrations. The results are shown in Figs 2 and 3.
Evaluation of the inhibitory effect of dentine powder on the antibacterial activity of the medicaments Dentine powder had a variable inhibitory effect, ranging from very slight to strong, after 1 h incubation period on the medicaments dependent on their concentration. NaOCl and PC Ag NPs were not inhibited by dentine at any concentration used after 24 h. The survival of E. faecalis for full strength NC Ag NPs was 65% and 30% in the absence of dentine during 1 and 24 h incubation which increased to 95% and 65% in the presence of dentine (P = 0.018). The results are summarized in Table 3.
Cytotoxicity of the medicaments on L929 fibroblasts Table 4 demonstrates the mean cell viability of L929 fibroblasts in each group after 1, 4 and 24 h exposure times. Repeated measures MANOVA test detected the presence of significant interaction effect (P < 0.001); denoting the dissimilar change in the mean cell viability of the groups over different exposure times. One-way ANOVA/Tukey tests demonstrated significant differences between the groups for each exposure time (P < 0.001). CHX was the most toxic solution to L929 fibroblasts (comparable to 35% H2O2) at all exposure times (P < 0.001). Cytotoxicity of PC Ag NPs in all three concentrations was significantly lower than CHX and NaOCl (P < 0.001).
90
2.4 9 10 3.7 9 10 5.7 9 10 _ _
3 3 10
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Discussion The recent increasing trend to use Ag NPs in root canal treatment reinforces the need for further assessment of its potential to be used as a root canal
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Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
(a)
(b)
(c)
Figure 2 Survival of E. faecalis when treated with different dilutions of negative (a), positive (b) and neutral (c) Ag NPs.
disinfectant. In this study, three types of Ag NPs were synthesized with positive, negative and neutral surface charges to assess their antimicrobial activity
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against planktonic E. faecalis compared with that of NaOCl and CHX. The second phase evaluated the influence of dentine powder on the inactivation of
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
Figure 3 Survival of E. faecalis when treated with different concentrations of the CHX and NaOCl.
these irrigants, and in the third phase, their cytotoxic effect against L929 mouse fibroblasts was determined. A strain of E. faecalis isolated from a root filled tooth with persistent apical periodontitis was used, given this pathogen is found with high prevalence in treated root canals and it is resistant to traditional root canal disinfectants (Evans et al. 2002, Stuart et al. 2006). NaOCl and CHX were used for all comparisons since they are the two most widely used irrigants amongst the numerous irrigation solutions used in endodontics. All solutions were examined in several concentrations as their antibacterial and cytotoxic effects are concentration dependent. Concentrations of Ag NPs were calculated according to the procedure suggested by Zhang et al. (2009a), using the size and absorbance of NPs, and reported in the unit of molar (mol L 1). This method was implemented because it was simple and
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
nondestructive. Other common strategies are (i) precipitating the NPs and then dispersing a weighed amount in the desired solvent, and (ii) using elemental analysis methods such as inductively coupled plasma (ICP) emission spectroscopy. The MIC results indicated that Ag NPs with positive surface charge were active in very low concentrations against planktonic E. faecalis compared to the other medicaments. In accordance with the general findings of the current study, Lotfi et al. (2013) demonstrated that a 70-fold concentration of NaOCl was required to achieve an antibacterial activity equal to Ag NPs. Although the MIC50 was defined for all medicaments, the MIC90 could not be determined for neutral and negative Ag NPs because they were not efficient at killing 90% of E. faecalis even at their initial concentrations. The difference in antimicrobial activity of the NPs may be a result of their surface charge and particle size. The carboxyl, phosphate and amino
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8
0.011 0 2% 0.33% 0.001
100% 10% 1.2% 40% 3.7%
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1 0 0 1 0.022 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 0.043 0.02% 0.007% 0.5% 0.23% 0.022 0.002 0 0 1 1 0 0 1 P-value 24 h 24 h* P-value
1 : 1000 1 : 100
100% 0 2% 0.48% 0.001 0 0 1 100% 0 0
1 : 10 1 : 100000
100% 0.01% 0.003% 0.4% 0.15%
100% 0 0.02% 0.036% 0.027 0 0 1
1 : 10000 1 : 1000
100% 0 0 100% 0 0
1 : 100 0.00025%
100% 0 0.01% 0.005% 0.010 0 0 1 100% 0 0
0.0025% 0.025%
100% 0 0 100% 0 0
0.25% 0.002%
100% 0.8% 0.2% 4% 0.93% 100% 0.01% 0.002% 0.3% 0.07%
0.02% 0.2%
100% 0 0 0h 1h 1 h*
Neut Ag NPs PC Ag NPs NaOCl CHX
Table 3 Survival of E. faecalis (mean SD) when treated with the selected concentrations/dilutions of the tested medicaments for 1 and 24 h in the absence and presence of dentin powder (*indicates the presence of dentin powder)
Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
groups on the cellular membrane of gram-positive bacteria dictate the negative charge of the organisms (van der Wal et al. 1997). Also, the nature of imidazole, used as a stabilizer in the synthesis process of PC Ag NPs, has indicated the positive surface charge and chemical functionality of NPs. It may be noted that the zeta potential or the surface charge of NPs may also strongly influence their stability in suspension through electrostatic repulsion between particles. Thus, the positive charge of ionic liquid-protected Ag NPs could interact with the negatively charged microbial cells and lead to altered cell wall permeability, formation of proteinaceous pores and leakage of intracellular components, which results in the extermination of E. faecalis. Besides, the affinity of PC Ag NPs to sulphur- and phosphorus-containing proteins of bacteria may potentially be a reason for differing antibacterial activity amongst Ag NPs (Le et al. 2011, 2012). However, further investigations are needed to interpret the exact mechanism. The results from the current study suggest the PC Ag NPs as a promising root canal disinfectant not only for their strong bactericidal potentials against E. faecalis but also for the limited effect of dentine on their antibacterial effectiveness together with their cytocompatibility, especially in low concentrations. The study demonstrated that PC Ag NPs at concentrations of 5.7 9 10 8 to 5.7 9 10 10 mol L 1 completely prevented the growth of E. faecalis in planktonic suspension after 5 min whilst the concentrations of 5.7 9 10 11 and 5.7 9 10 12 mol L 1 killed E. faecalis during 20 min of exposure. The study also demonstrated that E. faecalis did not survive after 4 and 24 h of contact time with the most diluted form of PC Ag NPs. Consistent with the findings of this study, another investigation established that 0.1% Ag NPs had a strong bactericidal effect against E. faecalis biofilm formed on dentine after 24 h of exposure (Zhuang et al. 2011). Furthermore, another study revealed that Ag NPs were able to destroy E. faecalis biofilm structures on dentine after an optimum period of interaction (Wu et al. 2014). Given it is established that tissue inhibitors may have a negative effect on root canal disinfectants (Haapasalo et al. 2000, Portenier et al. 2001, 2006), it is highly relevant to assess the impact of dentine on a newly introduced antimicrobial agent. Currently, no studies have evaluated the effect of tissue inhibitors in Ag NPs activity. However, a recent report on chitosan NPs indicated that dentine, dentine matrix and
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Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
Table 4 Mean SD of the cell viability (%) in the experimental groups at the defined exposure time Exposure time 35% H2O2 0.2% CHX 2.5% NaOCl PC Ag NPs (5.7 9 10 PC Ag NPs (5.7 9 10 PC Ag NPs (5.7 9 10
1h
8
mol L 1) mol L 1) 10 mol L 1) 9
5.52 11.57 65.55 91.20 94.51 101.41
4h A
1.75 2.14A 4.80B 1.95C 5.31C 1.92C
1.03 4.40 31.68 90.49 96.19 101.17
24 h A
0.67 1.42A 2.09B 3.39C 5.69CD 3.62D
0.18 2.07 11.21 80.70 95.36 103.47
0.31A 0.40A 2.09B 0.92C 3.35D 3.55E
Read vertically, same letters or having a letter in common, indicate lack of statistically significant difference (P < 0.05).
bacterial lipopolysaccharides did not influence the antibacterial activity of chitosan NPs (Shrestha & Kishen 2012). The current study discovered that the effect of dentine in inactivation of PC Ag NPs was extremely limited and was both time and concentration dependent. The presence of dentine powder had slight inhibitory effect on the antibacterial activity PC Ag NPs after 1 h of contact time at concentrations of 5.7 9 10 12 and 5.7 9 10 13 mol L 1 (1 : 10000 and 1 : 100000 dilutions), but it did not affect NPs activity after 24 h at the other tested concentrations. Ag NPs are frequently considered as being highly effective as antimicrobial agents whilst being nontoxic to mammals. In the present study, an L929 cell line was used, given it is a well-characterized cell model and has been previously used to assess the cytotoxic effects of dental materials (Faria et al. 2007, Ashraf et al. 2012). As demonstrated in Table 4, the medicaments inhibited the viability of fibroblasts in a timedependent manner. Amongst the medicaments tested, 0.2% CHX was the most cytotoxic agent to L929 fibroblasts at all tested exposure times and inhibited cell viability as much as the positive control (35% H2O2). These high grades of CHX cytotoxicity have been demonstrated previously (Faria et al. 2007). Referring to the results, it was established that PC Ag NPs at a concentration of 5.7 9 10 10 mol L 1 seem to be a cytocompatible solution as it did not affect cell viability by extending the exposure time from 1 h to 24 h. Likewise, a report by Gomes-Filho et al. (2010) concluded that 47 ppm nanosilver dispersions were biocompatible with fibrous connective tissue of rats compared to 2.5% NaOCl solution. Although various cytotoxicity studies have been performed on biological aspects of Ag NPs and have found that oxidative stress, damage to cellular components and cytokine induction were the main mechanisms of their cytotoxicity (Ahamed et al. 2008, Carlson et al. 2008, Hsin et al. 2008), the clear mechanism is still unknown. It has been demonstrated that a
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combination of physical and chemical properties of NPs including shape, size and surface charge together with their interactions in biological systems may affect NPs behaviour and determine their toxicity (Oberdorster et al. 2005, Maynard et al. 2011). Furthermore, stabilizing agents that bind to the entire NPs surface and inhibit their aggregation can also affect the cytotoxic behaviour of NPs. The imidazolium salt which was used as a stabilizing agent in the synthesis of PC Ag NPs is frequently known as a room temperature ionic liquid and previously has been employed as a green solvent for organic synthesis (Ryu et al. 2008). Imidazole-based polymeric micro and nano particles have also been synthesized and demonstrated to have little cytotoxicity (Jana et al. 2009, Zhang et al. 2009b). In the same way, the results demonstrated that Ag NPs protected by imidazole had little cytotoxicity to L929 fibroblasts whilst they were very active against E. faecalis. Although Ag NPs can enter both prokaryotic and eukaryotic cells, it seems that cellular antioxidant mechanisms in eukaryotic cells may protect them from possible oxidative damage (Arora et al. 2009). The other hypothesis of this finding may also be explained by the difference in the amount of particle uptake between different cell types.
Conclusion All three null hypotheses were rejected. This experimental investigation demonstrated the potential advantage of using positively charged ionic liquid-protected Ag NPs against E. faecalis where dentine had little inhibitory effect on their antibacterial activity. It was also established that these particles possessed a considerably lower cytotoxic effect against in vitro L929 fibroblast cell lines when compared with NaOCl and CHX. Furthermore, the importance of charge on the NPs surface and its role in bactericidal activity against E. faecalis were highlighted.
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Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
Acknowledgements The authors thank the Vice-Chancellery of Shiraz University of Medical Sciences. Also, we would like to thank Dr. Shahram Hamedani and Dr. Mehrdad Vosooghi for their valuable comments to improve the quality of the paper.
References Ahamed M, Karns M, Goodson M et al. (2008) DNA damage response to different surface chemistry of silver nanoparticles in mammalian cells. Toxicology and applied pharmacology 233, 404–10. Arora S, Jain J, Rajwade JM, Paknikar KM (2009) Interactions of silver nanoparticles with primary mouse fibroblasts and liver cells. Toxicology and applied pharmacology 236, 310–8. Ashraf H, Moradimajd N, Mozayeni MA, Dianat O, Mahjour F, Yadegari Z (2012) Cytotoxicity evaluation of three resin-based sealers on an L929 cell line. Dental Research Journal 9, 549–53. Carlson C, Hussain SM, Schrand AM et al. (2008) Unique cellular interaction of silver nanoparticles: size-dependent generation of reactive oxygen species. The Journal of Physical Chemistry B 112, 13608–19. Chaloupka K, Malam Y, Seifalian AM (2010) Nanosilver as a new generation of nanoproduct in biomedical applications. Trends in biotechnology 28, 580–8. Evans M, Davies JK, Sundqvist G, Figdor D (2002) Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. International Endodontic Journal 35, 221–8. Faria G, Celes MR, De Rossi A, Silva LA, Silva JS, Rossi MA (2007) Evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured L929 fibroblasts. Journal of Endodontics 33, 715–22. Gomes-Filho JE, Silva FO, Watanabe S et al. (2010) Tissue reaction to silver nanoparticles dispersion as an alternative irrigating solution. Journal of Endodontics 36, 1698– 702. Greenwood R, Kendall K (1999) Selection of suitable dispersants for aqueous suspensions of zirconia and titania powders using acoustophoresis. Journal of the European Ceramic Society 19, 479–88. Haapasalo HK, Siren EK, Waltimo TM, Orstavik D, Haapasalo MP (2000) Inactivation of local root canal medicaments by dentine: an in vitro study. International Endodontic Journal 33, 126–31. Hemmateenejad B, Dorostkar S, Shakerizadeh-Shirazi F, Shamsipur M (2013) pH-independent optical sensing of heparin based on ionic liquid-capped gold nanoparticles. The Analyst 138, 4830–7. Hsin YH, Chen CF, Huang S, Shih TS, Lai PS, Chueh PJ (2008) The apoptotic effect of nanosilver is mediated by a
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ROS- and JNK-dependent mechanism involving the mitochondrial pathway in NIH3T3 cells. Toxicology Letters 179, 130–9. Jana NR, Patra PK, Saha A, Basiruddin S, Pradhan N (2009) Imidazole based biocompatible polymer coating in deriving< 25 nm functional nanoparticle probe for cellular imaging and detection. The Journal of Physical Chemistry C 113, 21484–92. Kim JS, Kuk E, Yu KN et al. (2007) Antimicrobial effects of silver nanoparticles. Nanomedicine 3, 95–101. Kishen A, Shi Z, Shrestha A, Neoh KG (2008) An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. Journal of Endodontics 34, 1515–20. Le A-T, Huy PT, Tam LT, Tam PD, Hieu N, Huy T (2011) Novel silver nanoparticles: synthesis, properties and applications. International Journal of Nanotechnology 8, 278–90. Le A-T, Le TT, Tran HH, Dang DA, Tran QH, Vu DL (2012) Powerful colloidal silver nanoparticles for the prevention of gastrointestinal bacterial infections. Advances in Natural Sciences: Nanoscience and Nanotechnology 3, 045007. Lotfi M, Vosoughhosseini S, Ranjkesh B, Khani S, Saghiri M, Zand V (2013) Antimicrobial efficacy of nanosilver, sodium hypochlorite and chlorhexidine gluconate against Enterococcus faecalis. African Journal of Biotechnology 10, 6799–803. Maynard AD, Warheit DB, Philbert MA (2011) The new toxicology of sophisticated materials: nanotoxicology and beyond. Toxicological Sciences 120(Suppl 1), S109–29. Oberdorster G, Oberdorster E, Oberdorster J (2005) Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environmental Health Perspectives 113, 823–39. Portenier I, Haapasalo H, Rye A, Waltimo T, Orstavik D, Haapasalo M (2001) Inactivation of root canal medicaments by dentine, hydroxylapatite and bovine serum albumin. Intennatioanl Endodontic Journal 34, 184–8. Portenier I, Waltimo T, Orstavik D, Haapasalo M (2006) Killing of Enterococcus faecalis by MTAD and chlorhexidine digluconate with or without cetrimide in the presence or absence of dentine powder or BSA. Journal of Endodontics 32, 138–41. Rai MK, Deshmukh SD, Ingle AP, Gade AK (2012) Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. Journal of Applied Microbiology 112, 841–52. Ryu HJ, Sanchez L, Keul HA, Raj A, Bockstaller MR (2008) Imidazolium-based ionic liquids as efficient shape-regulating solvents for the synthesis of gold nanorods. Angewandte Chemie International Edition 47, 7639–43. Shrestha A, Kishen A (2012) The effect of tissue inhibitors on the antibacterial activity of chitosan nanoparticles and photodynamic therapy. Journal of Endodontics 38, 1275–8. Shrestha A, Shi Z, Neoh KG, Kishen A (2010) Nanoparticulates for antibiofilm treatment and effect of aging on its antibacterial activity. Journal of Endodontics 36, 1030–5.
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Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
Shrivastava S, Bera T, Roy A, Singh G, Ramachandrarao P, Dash D (2007) Characterization of enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology 18, 225103. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB (2006) Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. Journal of Endodontics 32, 93–8. Tran QH, Le A-T (2013) Silver nanoparticles: synthesis, properties, toxicology, applications and perspectives. Advances in Natural Sciences: Nanoscience and Nanotechnology 4, 033001. van der Wal A, Norde W, Zehnder AJ, Lyklema J (1997) Determination of the total charge in the cell walls of Gram-positive bacteria. Colloids and surfaces B: Biointerfaces 9, 81–100. Wikler MA (2010) Performance Standards for Antimicrobial Susceptibility Testing:Twentieth Informational Supplement. Wayne, PA: Clinical and Laboratory Standards Institute.
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Wu D, Fan W, Kishen A, Gutmann JL, Fan B (2014) Evaluation of the antibacterial efficacy of silver nanoparticles against Enterococcus faecalis biofilm. Journal of Endodontics 40, 285–90. Zandi-Atashbar N, Hemmateenejad B, Akhond M (2011) Determination of amylose in Iranian rice by multivariate calibration of the surface plasmon resonance spectra of silver nanoparticles. The Analyst 136, 1760–6. Zhang J, Fu Y, Liang D, Zhao RY, Lakowicz JR (2009a) Fluorescent avidin-bound silver particle: a strategy for single target molecule detection on a cell membrane. Analytical Chemistry 81, 883–9. Zhang Y, Zhao L, Patra PK, Hu D, Ying JY (2009b) Colloidal poly-imidazolium salts and derivatives. Nano Today 4, 13–20. Zhuang P, Gao Y, Ling J, Hu X, Wang Z (2011) Bactericidal effect of nano-silver against E. faecalis biofilm on dentin. Chinese Journal of Stomatological Research Electronic Edition 5, 463–9.
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Positively charged imidazolium-based ionic liquid-protected silver nanoparticles: a promising disinfectant in root canal treatment
A. Abbaszadegan1, M. Nabavizadeh1, A. Gholami2, Z. S. Aleyasin3, S. Dorostkar4, M. Saliminasab5, Y. Ghasemi2, B. Hemmateenejad4 & H. Sharghi4 1
Department of Endodontics, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz; 2Department of Pharmaceutical Biotechnology and Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz; 3Department of Oral Medicine, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz; 4Department of Chemistry, Shiraz University, Shiraz; and 5Shiraz Dental School, Shiraz, Iran
Abstract Abbaszadegan A, Nabavizadeh M, Gholami A, Aleyasin ZS, Dorostkar S, Saliminasab M, Ghasemi Y, Hemmateenejad B, Sharghi H. Positively charged imidazolium-based ionic liquid-protected silver nanoparticles: a promising disinfectant in root canal treatment. International Endodontic Journal.
Aim To synthesize and characterize silver nanoparticles (Ag NPs) with different surface charges in order to evaluate their cytotoxicity and antibacterial activity in the absence and presence of dentine compared with NaOCl and CHX. Methodology Ag NPs with positive, negative and neutral surface charges were synthesized and characterized. The first phase of the experiment determined the minimum inhibitory concentrations (MICs) of NPs against planktonic E. faecalis and compared them with that of NaOCl and CHX. The second phase tested the elimination of E. faecalis at different contact times (5, 20 and 60 min and 4 and 24 h), and the role of dentine in their inactivation was assessed. In the third phase, the most effective Ag NP solution was selected for cytocompatibility assessment. An MTT-based cytotoxicity assay was used to evaluate the cytotoxicity of the selected NP solution in different concentrations on L929 fibroblasts compared to that of
2.5% NaOCl and 0.2% CHX. Student’s t-test and repeated measures MANOVA approach were used for statistical analyses. Results The characterization revealed synthesis of colloidal NPs in the size range of 5–10 nm in diameter. The results indicated that Ag NP with a positive surface charge had the smallest MIC against planktonic E. faecalis, and it was active in very lower concentrations compared to NaOCl, CHX and the other tested AgNPs. Positive-charged Ag NPs at 5.7 9 10 10 mol L 1 completely prevented the growth of E. faecalis after 5 min of contact time, a finding comparable to 0.025% NaOCl. Dentine powder had variable inhibitory effects on all tested materials after 1 h incubation period, but after 24 h, NaOCl and the positive-charged Ag NPs were not inhibited by dentine at any concentration used. CHX was the most and the positively charged Ag NP solution was the least toxic solutions to L929 fibroblasts (P < 0.001). Conclusions Ag NP surface charge was important in bactericidal efficacy against E. faecalis. The positively charged imidazolium-based ionic liquidprotected Ag NPs showed promising antibacterial results against E. faecalis and exhibited a high level of cytocompatibility to L929 cells. Keywords: antimicrobial, cytotoxicity, dentin, Enterococcus faecalis, ionic liquid, silver nanoparticles. Received 24 February 2014; accepted 27 August 2014
Correspondence: Mohammadreza Nabavizadeh, Ghomabad St, Ghasrodasht St, Dental School, Shiraz, Iran (Tel.: +989173029846; Fax: +987116263192; e-mail: nabaviza [email protected]).
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
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Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
Introduction Nanotechnology continues to have a critical impact in all disciplines including dental sciences. Consequently, endodontics can benefit from this field of research due to the various chemical and physical properties of the materials produced on a nanoscale. Recent studies have reported the strong antibacterial activity of nanoparticles such as chitosan and zinc oxide against Enterococcus faecalis (Kishen et al. 2008, Shrestha et al. 2010). Silver nanoparticles (Ag NPs) have also drawn the attention of researchers in recent endodontic investigations. The antibacterial activity of Ag NPs against several species of endodontic bacteria has been recorded in several investigations (Kim et al. 2007, Shrivastava et al. 2007, Lotfi et al. 2013, Wu et al. 2014). Besides, antibacterial nanoparticles do not seem to provide the bacteria with the capacity to gain resistance (Rai et al. 2012). Therefore, they may serve as promising disinfectants for endodontic purposes. Nanoparticles (NPs) can be synthesized by various methods which influence the particle’s characteristics including morphology, surface charge, stability and chemical reactivity (Chaloupka et al. 2010). The antimicrobial properties of several metallic NPs are well documented. This feature may be influenced by variables including the size, shape and the surface charge of the particles (Tran & Le 2013). Also, the same variables play a substantial role in nanotoxicity (Tran & Le 2013). There is no comprehensive study in the literature to assess the cytotoxicity and antimicrobial activity of Ag NPs with different surface charges against E. faecalis relative to other endodontic irrigants to enlighten the position of these particles in future endodontic practice. Likewise, reports focusing on the surface charge of Ag NPs and its relevance to antimicrobial effectiveness have proven inconclusive. In this study, stable Ag NPs with different sizes and surface charges were synthesized and characterized. The antibacterial activity and cytotoxicity of these products were compared with sodium hypochlorite (NaOCl) and chlorhexidine (CHX), and the role of dentine in inactivation of these irrigants was assessed. The null hypotheses tested were as follows: (i) there is no difference in antibacterial efficacy of differently charged Ag NPs against planktonic E. faecalis when compared with NaOCl and CHX, (ii) dentine has no inhibitory effect on antibacterial activity of these irrigants, and (iii) there is no difference amongst the
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cytocompatibilities of these irrigants against L929 mouse fibroblasts.
Materials and methods Synthesis of neutral silver NPs The neutral Ag NPs (Neut Ag NPs) were synthesized using the method described by Zandi-Atashbar et al. (2011) in which the milled rice samples were used directly with no sample pre-processing. Powdered rice samples were dried by storing at 60 °C overnight. A 100.0 mg portion of the rice powders was added into a test tube containing 10.0 mL buffer solution whilst the tubes were placed in a boiling water bath for 15 min. Furthermore, 1.0 mL of AgNO3 (0.01 molar) was added, and the resulting suspension was heated for an additional 5 min, until the colour of the solution became golden.
Synthesis of negative-charged silver NP Negative-charged Ag NPs (NC Ag NPs) were synthesized through the reduction of silver nitrate using sodium borohydride. All glassware was placed in 1 : 3 HCl/HNO3 solution and then rinsed with triple distilled water for three times. Furthermore, the sodium borohydride was chilled to roughly 0 °C using ice and stirring. A 1.0 mL portion of 0.01 m mol L 1 AgNO3 was added to 20 mL of the stirring 6.2 m mol L 1 NaBH4 solution using a dropper, at approximately one drop per second. Stirring was stopped after complete addition of AgNO3.
Synthesis of positive-charged silver NPs The ionic liquid-protected silver nanoparticles (PC Ag NPs) were prepared according to the procedure described by Hemmateenejad et al. (2013). In brief; all glassware was placed in 1 : 3 HCl/HNO3 solution, rinsed with triple distilled water three times; then, 1.0 mL of 0.01 mol L 1 AgNO3 aqueous solution was added to 20 mL of 6.2 m mol L 1 1-dodecyl-3-methylimidazolium chloride ([C12mim][Cl]) aqueous solution, and the solution was stirred vigorously. The freshly prepared 0.4 mol L 1 NaBH4 aqueous solution was then added to the stirred solutions dropwise until the colour of the solution became golden. Subsequently, the colloidal solutions were centrifuged for 20 min to remove excess amount of ionic liquids. The resultant goldencoloured solution was stored at room temperature.
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
Characterization of Ag NPs As the NP characterization is necessary to establish the understanding and control of NP synthesis and application, the formation of Ag NPs was evidenced by UV–visible spectra of the colloidal silver solutions. Absorption spectra were recorded on an Ultrospec 3000 UV–vis spectrophotometer (Pharmacia Biotech, San Francisco, CA, USA) equipped with a 1 cm quartz cell. Furthermore, to have a better estimation of the size range of the prepared Ag NPs, they were characterized with transmission electron microscopy. The measurements were made on a transmission electron microscope (TEM, JEM 2010, Jeol, Tokyo, Japan) operated at an accelerating voltage of 100 kV. Samples for TEM characterization were prepared by placing a drop of the as-prepared silver colloidal samples (wellcentrifuged samples) on a carbon-coated copper grid and then drying at room temperature. Several TEM images were prepared for each synthesized Ag NP, and the average size of 250 particles in these images was recorded. Using the size and absorbance of the NPs, the concentrations of the aqueous solution of the synthesized NPs were calculated according to the procedure suggested by Zhang et al. (2009a). The surface charge or zeta potential of the synthesized NPs was verified by zeta potential analysis using zeta potential analyser (Zeta Plus, Brookhaven Instrument Co., Holtsville, NY, USA).
Measurement of antibacterial activity against E. faecalis Medicaments The experimental groups were consisted of negative, neutral and positive-charged Ag NPs at their initial synthesized concentrations, 2.5% NaOCl (SigmaAldrich Co., St. Louis, MO, USA) and 0.2% CHX (Sigma-Aldrich Co.). All medicaments were then diluted from their initial concentrations. Sterile water was used as negative control. All medicaments were freshly prepared prior to commencement of experiments. Test organism The test organism in this study was E. faecalis strain AGH 011, isolated from a previously root filled tooth with persistent apical periodontitis (GeneBank: KF701470). The strain was identified by gram
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
staining, catalase reaction, colony morphology and by the patterns of carbohydrate fermentation and enzyme production. An overnight culture on TSA plates (Tryptic-soy-agar, Difco, MI, USA) was checked for purity, suspended in 0.5% peptone water (Bacto peptone, Difco) and adjusted spectrophotometrically to match the turbidity of 2 9 109 CFU mL 1. Minimum inhibitory concentration (MIC) determination MIC determination of each medicament against E. faecalis was conducted three times (triplicate) by a microdilution method with Mueller-Hinton broth (Himedia, Mumbai, India) supplemented with calcium (25 mg L 1) and magnesium (12.5 mg L 1). All procedures were performed according to the guidelines presented by the Clinical and Laboratory Standards Institute (CLSI) (Wikler 2010). 10-fold serial dilutions of each medicament were prepared (up to eight times). Then, the MIC50 and MIC90 were defined as the lowest concentration, which inhibited 50% and 90% of the growth when compared with growth control, respectively. Elimination of E. faecalis in different contact times For the first phase of the experiment, 950 lL of each experimental solution was mixed with 50 lL of bacterial suspension in an Eppendorf test tube. In this step, imidazole solution was also employed as an experimental solution because of its role in PC Ag NPs synthesis. These suspensions were carefully mixed and incubated at 37 °C in air. Furthermore, 10 lL of each sample was collected after determined incubation periods (5, 20 and 60 min and 4 and 24 h). Each sample was utilized for preparing 10-fold serial dilutions (up to five times). Each dilution was cultured on a TSA plate at 37 °C for 24 h, and following, the plates were checked for the bacterial growth under a stereomicroscope. The number of colonies was calculated, and the bacterial growth was measured by the CFU mL 1 counts of E. faecalis. Microscopic observation of the colonies was carried out to rule out any contamination. These procedures were repeated for three times to reduce the possible bias.
Evaluation of the inhibitory effect of dentine powder on the antibacterial activity of the medicaments This protocol was adopted from Portenier et al. (2006). Freshly extracted human third molar teeth were collected and kept in 0.5% NaOCl for 1 h to
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Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
clear soft tissue residues and to stop microbial growth. The teeth were rinsed with distilled water and then autoclaved at 121 °C for 15 min. The roots were separated from the crowns and sectioned to produce small slices using a low-speed diamond saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA). The root slices were then grounded by a planetary ball-milling device (Retschâ PM400, Retsch GmbH, Haan, Germany) at 400 rpm for 6 h to yield the dentine powder with a particle size of 0.2–20 lm in diameter. Selected concentrations of the medicaments were employed to include in this part of study based on data obtained from their effectiveness in the absence of dentine. In an Eppendorf tube, per experiment, a total of 28 mg of dentine powder was suspended in 50 lL sterile water, then, carefully mixed with 50 lL of the bacterial suspension (1.0 9 108 CFU) together with 50 lL of each medicaments (total volume: 150 lL). In control tubes, dentine powder was not used and another 50 lL of sterile water was added to keep the total volume of 150 lL. The suspensions were incubated at 37 °C in an aerophilic environment for 1 h and 24 h. Furthermore, 10 lL of each sample was cultured, and the number of colonies was counted. This procedure was performed in triplicate.
sulphoxide (DMSO) was added to each well. The absorption of the solution was at a wavelength of 540 nm by an ELISA plate reader (PowerWaveTM X52, BioTek Instruments Inc., Potton, UK). This indirect cytotoxicity test was performed three times for each sample. The mean optical density values (OD) of three wells containing the same extract with their standard deviations were calculated. The mean cell viability values were defined as the percentage of the OD values of the negative control.
Statistical analysis The data obtained from the effectiveness of the experimental solutions, in the absence and presence of dentine, were evaluated for the statistical significance using the Student’s t-test. Repeated measures MANOVA approach and subgroup analysis were used to evaluate the differences in the mean cell viability values of L929 fibroblasts. For the purpose of subgroup analysis, one-way ANOVA/Tukey tests were used. A level of P < 0.05 was accepted as statistically significant.
Results Characterization of Ag NPs
Cytotoxicity of the medicaments on L929 fibroblasts Cytotoxicity of the medicaments on L929 mouse fibroblasts was evaluated using MTT colorimetric assay. Three different concentrations of PC Ag NPs ranging from 5.7 9 10 8 to 5.7 9 10 10 mol L 1, 2.5% NaOCl and 0.2% CHX were selected. Culture medium and 35% hydrogen peroxide were regarded as the negative and positive controls, respectively. L929 mouse fibroblasts in RPMI 1640 media were aliquoted into a 96-well cell culture plate at a density of approximately 1 9 104 cells/well and incubated in a humidified atmosphere of 5% CO2, 95% air at 37 °C to reach roughly 80% confluence. After 24 h, 100 lL of each sample which was previously incubated at 37 °C in RPMI 1640 media for 24 h was transferred into each well. The medium was removed at 1, 4, and 24 h after incubation, and the wells were washed twice for 2–3 min with 200 lL of the fresh medium. A total of 25 lL of MTT [3-(4,5 Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide] (Sigma-Aldrich Co.) stock solution was added to each well and incubated in a humidified atmosphere of 5% CO2, 95% air for 4 h at 37 °C. Then, 100 lL of dimethyl
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The peak of the plasmon resonance band of the synthesized Ag NPs was located at about 400 nm (Table 1) attributing to the formation of Ag NPs in the size ranges of 5–10 nm in diameter (Zhang et al. 2009a). The sample TEM images are given in Fig. 1, and the average sizes of the synthesized Ag NPs, based on the TEM images, and their initial concentrations are listed in Table 1. The mean particle sizes measured based on particle counting were in good agreement with those estimated by absorbance peak of the surface plasmon resonance bands. However, for the Neut Ag NPs, an overlap between absorbance of starch and surface plasmon resonance of Ag NPs made the estimation of the net absorbance contribution of Ag NPs impractical. Consequently, determination of the exact concentration of Ag NPs was unfeasible. The zeta Table 1 Properties of the synthesized Ag NPs Surface charge
kmax (nm)
Absorbancemax (nm)
Average size (nm)
Negative Neutral Positive
400 425 407
0.45 ND 1.8
7.5 10.1 9.0
Concentration (mol L 1) 9.7 9 10 4.0 9 10 5.7 9 10
8 8 8
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
(a)
(b)
(c)
Figure 1 Sample TEM images of negative (a), positive (b) and neutral (c) Ag NPs.
potential of the NC Ag NPs was obtained as -38.0 mV, whereas the surface charges of the synthesized PC Ag NPs and Neut Ag NPs were determined as +50.0 mV and 0.0 mV, respectively. These values of zeta potential represent a good stability for the charged NPs (Greenwood & Kendall 1999).
Minimum inhibitory concentration determination Table 2 demonstrates the MIC determination for E. faecalis when exposed to each tested medicament.
Elimination of E. faecalis at different contact times Enterococcus faecalis had 100% growth when treated with sterile water as control. For the experimental groups, the survival rate of E. faecalis was expressed as a percentage of survival in the control group. Different concentrations of imidazole showed 100% growth for E. faecalis in all defined incubation periods. The antibacterial effect of other solutions was dependent on their concentration and contact time. 0.2% CHX completely inhibited the growth of E. faecalis at all incubation periods. PC Ag NPs at 5.7 9 10 10 mol L 1 and NaOCl at 0.025% completely prevented the growth of E. faecalis even after 5 min of contact time. NC Ag NPs at full strength concentration (9.7 9 10 8 mol L 1) started to inhibit the growth of E. faecalis after 1 h of contact time. Neut Ag NPs had moderate inhibitory effect on E. fae-
Table 2 The minimum inhibitory concentrations (mol L
1
)
of the tested medicaments against E. faecalis Medicaments
MIC
CHX NaOCl PC Ag NPs NC Ag NPs Neut Ag NPs
9 9 9 9 9
2.4 3.7 5.7 9.7 4.0
MIC
50
10 10 10 10 10
5 7 15 8 9
calis dependent on their concentrations. The results are shown in Figs 2 and 3.
Evaluation of the inhibitory effect of dentine powder on the antibacterial activity of the medicaments Dentine powder had a variable inhibitory effect, ranging from very slight to strong, after 1 h incubation period on the medicaments dependent on their concentration. NaOCl and PC Ag NPs were not inhibited by dentine at any concentration used after 24 h. The survival of E. faecalis for full strength NC Ag NPs was 65% and 30% in the absence of dentine during 1 and 24 h incubation which increased to 95% and 65% in the presence of dentine (P = 0.018). The results are summarized in Table 3.
Cytotoxicity of the medicaments on L929 fibroblasts Table 4 demonstrates the mean cell viability of L929 fibroblasts in each group after 1, 4 and 24 h exposure times. Repeated measures MANOVA test detected the presence of significant interaction effect (P < 0.001); denoting the dissimilar change in the mean cell viability of the groups over different exposure times. One-way ANOVA/Tukey tests demonstrated significant differences between the groups for each exposure time (P < 0.001). CHX was the most toxic solution to L929 fibroblasts (comparable to 35% H2O2) at all exposure times (P < 0.001). Cytotoxicity of PC Ag NPs in all three concentrations was significantly lower than CHX and NaOCl (P < 0.001).
90
2.4 9 10 3.7 9 10 5.7 9 10 _ _
3 3 10
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Discussion The recent increasing trend to use Ag NPs in root canal treatment reinforces the need for further assessment of its potential to be used as a root canal
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Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
(a)
(b)
(c)
Figure 2 Survival of E. faecalis when treated with different dilutions of negative (a), positive (b) and neutral (c) Ag NPs.
disinfectant. In this study, three types of Ag NPs were synthesized with positive, negative and neutral surface charges to assess their antimicrobial activity
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against planktonic E. faecalis compared with that of NaOCl and CHX. The second phase evaluated the influence of dentine powder on the inactivation of
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
Figure 3 Survival of E. faecalis when treated with different concentrations of the CHX and NaOCl.
these irrigants, and in the third phase, their cytotoxic effect against L929 mouse fibroblasts was determined. A strain of E. faecalis isolated from a root filled tooth with persistent apical periodontitis was used, given this pathogen is found with high prevalence in treated root canals and it is resistant to traditional root canal disinfectants (Evans et al. 2002, Stuart et al. 2006). NaOCl and CHX were used for all comparisons since they are the two most widely used irrigants amongst the numerous irrigation solutions used in endodontics. All solutions were examined in several concentrations as their antibacterial and cytotoxic effects are concentration dependent. Concentrations of Ag NPs were calculated according to the procedure suggested by Zhang et al. (2009a), using the size and absorbance of NPs, and reported in the unit of molar (mol L 1). This method was implemented because it was simple and
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
nondestructive. Other common strategies are (i) precipitating the NPs and then dispersing a weighed amount in the desired solvent, and (ii) using elemental analysis methods such as inductively coupled plasma (ICP) emission spectroscopy. The MIC results indicated that Ag NPs with positive surface charge were active in very low concentrations against planktonic E. faecalis compared to the other medicaments. In accordance with the general findings of the current study, Lotfi et al. (2013) demonstrated that a 70-fold concentration of NaOCl was required to achieve an antibacterial activity equal to Ag NPs. Although the MIC50 was defined for all medicaments, the MIC90 could not be determined for neutral and negative Ag NPs because they were not efficient at killing 90% of E. faecalis even at their initial concentrations. The difference in antimicrobial activity of the NPs may be a result of their surface charge and particle size. The carboxyl, phosphate and amino
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8
0.011 0 2% 0.33% 0.001
100% 10% 1.2% 40% 3.7%
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1 0 0 1 0.022 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 1 0 0 1 0.043 0.02% 0.007% 0.5% 0.23% 0.022 0.002 0 0 1 1 0 0 1 P-value 24 h 24 h* P-value
1 : 1000 1 : 100
100% 0 2% 0.48% 0.001 0 0 1 100% 0 0
1 : 10 1 : 100000
100% 0.01% 0.003% 0.4% 0.15%
100% 0 0.02% 0.036% 0.027 0 0 1
1 : 10000 1 : 1000
100% 0 0 100% 0 0
1 : 100 0.00025%
100% 0 0.01% 0.005% 0.010 0 0 1 100% 0 0
0.0025% 0.025%
100% 0 0 100% 0 0
0.25% 0.002%
100% 0.8% 0.2% 4% 0.93% 100% 0.01% 0.002% 0.3% 0.07%
0.02% 0.2%
100% 0 0 0h 1h 1 h*
Neut Ag NPs PC Ag NPs NaOCl CHX
Table 3 Survival of E. faecalis (mean SD) when treated with the selected concentrations/dilutions of the tested medicaments for 1 and 24 h in the absence and presence of dentin powder (*indicates the presence of dentin powder)
Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
groups on the cellular membrane of gram-positive bacteria dictate the negative charge of the organisms (van der Wal et al. 1997). Also, the nature of imidazole, used as a stabilizer in the synthesis process of PC Ag NPs, has indicated the positive surface charge and chemical functionality of NPs. It may be noted that the zeta potential or the surface charge of NPs may also strongly influence their stability in suspension through electrostatic repulsion between particles. Thus, the positive charge of ionic liquid-protected Ag NPs could interact with the negatively charged microbial cells and lead to altered cell wall permeability, formation of proteinaceous pores and leakage of intracellular components, which results in the extermination of E. faecalis. Besides, the affinity of PC Ag NPs to sulphur- and phosphorus-containing proteins of bacteria may potentially be a reason for differing antibacterial activity amongst Ag NPs (Le et al. 2011, 2012). However, further investigations are needed to interpret the exact mechanism. The results from the current study suggest the PC Ag NPs as a promising root canal disinfectant not only for their strong bactericidal potentials against E. faecalis but also for the limited effect of dentine on their antibacterial effectiveness together with their cytocompatibility, especially in low concentrations. The study demonstrated that PC Ag NPs at concentrations of 5.7 9 10 8 to 5.7 9 10 10 mol L 1 completely prevented the growth of E. faecalis in planktonic suspension after 5 min whilst the concentrations of 5.7 9 10 11 and 5.7 9 10 12 mol L 1 killed E. faecalis during 20 min of exposure. The study also demonstrated that E. faecalis did not survive after 4 and 24 h of contact time with the most diluted form of PC Ag NPs. Consistent with the findings of this study, another investigation established that 0.1% Ag NPs had a strong bactericidal effect against E. faecalis biofilm formed on dentine after 24 h of exposure (Zhuang et al. 2011). Furthermore, another study revealed that Ag NPs were able to destroy E. faecalis biofilm structures on dentine after an optimum period of interaction (Wu et al. 2014). Given it is established that tissue inhibitors may have a negative effect on root canal disinfectants (Haapasalo et al. 2000, Portenier et al. 2001, 2006), it is highly relevant to assess the impact of dentine on a newly introduced antimicrobial agent. Currently, no studies have evaluated the effect of tissue inhibitors in Ag NPs activity. However, a recent report on chitosan NPs indicated that dentine, dentine matrix and
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
Abbaszadegan et al. Toxicity and antibacterial activity of Ag nanoparticles
Table 4 Mean SD of the cell viability (%) in the experimental groups at the defined exposure time Exposure time 35% H2O2 0.2% CHX 2.5% NaOCl PC Ag NPs (5.7 9 10 PC Ag NPs (5.7 9 10 PC Ag NPs (5.7 9 10
1h
8
mol L 1) mol L 1) 10 mol L 1) 9
5.52 11.57 65.55 91.20 94.51 101.41
4h A
1.75 2.14A 4.80B 1.95C 5.31C 1.92C
1.03 4.40 31.68 90.49 96.19 101.17
24 h A
0.67 1.42A 2.09B 3.39C 5.69CD 3.62D
0.18 2.07 11.21 80.70 95.36 103.47
0.31A 0.40A 2.09B 0.92C 3.35D 3.55E
Read vertically, same letters or having a letter in common, indicate lack of statistically significant difference (P < 0.05).
bacterial lipopolysaccharides did not influence the antibacterial activity of chitosan NPs (Shrestha & Kishen 2012). The current study discovered that the effect of dentine in inactivation of PC Ag NPs was extremely limited and was both time and concentration dependent. The presence of dentine powder had slight inhibitory effect on the antibacterial activity PC Ag NPs after 1 h of contact time at concentrations of 5.7 9 10 12 and 5.7 9 10 13 mol L 1 (1 : 10000 and 1 : 100000 dilutions), but it did not affect NPs activity after 24 h at the other tested concentrations. Ag NPs are frequently considered as being highly effective as antimicrobial agents whilst being nontoxic to mammals. In the present study, an L929 cell line was used, given it is a well-characterized cell model and has been previously used to assess the cytotoxic effects of dental materials (Faria et al. 2007, Ashraf et al. 2012). As demonstrated in Table 4, the medicaments inhibited the viability of fibroblasts in a timedependent manner. Amongst the medicaments tested, 0.2% CHX was the most cytotoxic agent to L929 fibroblasts at all tested exposure times and inhibited cell viability as much as the positive control (35% H2O2). These high grades of CHX cytotoxicity have been demonstrated previously (Faria et al. 2007). Referring to the results, it was established that PC Ag NPs at a concentration of 5.7 9 10 10 mol L 1 seem to be a cytocompatible solution as it did not affect cell viability by extending the exposure time from 1 h to 24 h. Likewise, a report by Gomes-Filho et al. (2010) concluded that 47 ppm nanosilver dispersions were biocompatible with fibrous connective tissue of rats compared to 2.5% NaOCl solution. Although various cytotoxicity studies have been performed on biological aspects of Ag NPs and have found that oxidative stress, damage to cellular components and cytokine induction were the main mechanisms of their cytotoxicity (Ahamed et al. 2008, Carlson et al. 2008, Hsin et al. 2008), the clear mechanism is still unknown. It has been demonstrated that a
© 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd
combination of physical and chemical properties of NPs including shape, size and surface charge together with their interactions in biological systems may affect NPs behaviour and determine their toxicity (Oberdorster et al. 2005, Maynard et al. 2011). Furthermore, stabilizing agents that bind to the entire NPs surface and inhibit their aggregation can also affect the cytotoxic behaviour of NPs. The imidazolium salt which was used as a stabilizing agent in the synthesis of PC Ag NPs is frequently known as a room temperature ionic liquid and previously has been employed as a green solvent for organic synthesis (Ryu et al. 2008). Imidazole-based polymeric micro and nano particles have also been synthesized and demonstrated to have little cytotoxicity (Jana et al. 2009, Zhang et al. 2009b). In the same way, the results demonstrated that Ag NPs protected by imidazole had little cytotoxicity to L929 fibroblasts whilst they were very active against E. faecalis. Although Ag NPs can enter both prokaryotic and eukaryotic cells, it seems that cellular antioxidant mechanisms in eukaryotic cells may protect them from possible oxidative damage (Arora et al. 2009). The other hypothesis of this finding may also be explained by the difference in the amount of particle uptake between different cell types.
Conclusion All three null hypotheses were rejected. This experimental investigation demonstrated the potential advantage of using positively charged ionic liquid-protected Ag NPs against E. faecalis where dentine had little inhibitory effect on their antibacterial activity. It was also established that these particles possessed a considerably lower cytotoxic effect against in vitro L929 fibroblast cell lines when compared with NaOCl and CHX. Furthermore, the importance of charge on the NPs surface and its role in bactericidal activity against E. faecalis were highlighted.
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Toxicity and antibacterial activity of Ag nanoparticles Abbaszadegan et al.
Acknowledgements The authors thank the Vice-Chancellery of Shiraz University of Medical Sciences. Also, we would like to thank Dr. Shahram Hamedani and Dr. Mehrdad Vosooghi for their valuable comments to improve the quality of the paper.
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