Core-shell silver nanoparticles in endodontic disinfection solutions

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Core−Shell Silver Nanoparticles in Endodontic Disinfection Solutions Enable Long-Term Antimicrobial Effect on Oral Biofilms Elif Ertem,†,⊥ Beatrice Gutt,‡,⊥ Flavia Zuber,‡ Sergio Allegri,† Benjamin Le Ouay,§ Selma Mefti,∥ Kitty Formentin,∥ Francesco Stellacci,*,† and Qun Ren*,‡ †

Institute of Materials, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne CH 1015, Switzerland Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and Technology, St. Gallen CH 9014, Switzerland § Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan ∥ Dentsply Sirona, Ballaigues CH 1338, Switzerland ‡

S Supporting Information *

ABSTRACT: To achieve effective long-term disinfection of the root canals, we synthesized core−shell silver nanoparticles (AgNPs@SiO2) and used them to develop two irrigation solutions containing sodium phytate (SP) and ethylene glycol-bis(βaminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA), respectively. Ex vivo studies with instrumented root canals revealed that the developed irrigation solutions can effectively remove the smear layer from the dentinal surfaces. Further in vitro experiments with single- and multispecies biofilms demonstrated for the first time that AgNPs@SiO2based irrigation solutions possess excellent antimicrobial activities for at least 7 days, whereas the bare AgNPs lose the activity almost immediately and do not show any antibacterial activity after 2 days. The long-term antimicrobial activity exhibited by AgNPs@SiO2 solutions can be attributed to the sustainable availability of soluble silver, even after 7 days. Both solutions showed lower cytotoxicity toward human gingival fibroblasts compared to the conventionally used solution (3% NaOCl and 17% EDTA). Irrigation solutions containing AgNP@SiO2 may therefore be highly promising for applications needing a long-term antimicrobial effect. KEYWORDS: root-canal treatment, core−shell silver nanoparticles, irrigation solutions, oral biofilm, multispecies biofilm, antimicrobial activity ability to dissolve organic tissues.4 However, NaOCl is not effective in the removal of inorganic components from the smear layer, and thus a solution including a decalcifying agent such as ethylenediaminetetraacetic acid (EDTA), or citric or phosphoric acid should be used.5 This treatment is performed sequentially since the interactions between the two solutions cause a loss of NaOCl activity.5 Studies have shown that currently used irrigation media are only moderately effective and that bacteria still persist in significantly high numbers within the root canals after the treatment, which is one of the foremost reasons of endodontic failure.6,7 Furthermore, to ease and shorten the tooth treatment, all-in-one irrigation solutions instead of a sequential treatment are highly desired. The potential benefits of nanotechnology in the biomedical field have become widely accepted for the generation of promising strategies to treat various bacterial diseases.8 Silvercontaining (Ag-containing) nanomaterials are of extensive

1. INTRODUCTION The endodontic or root-canal treatment is a tooth-saving clinical practice that eliminates infected dental tissues and protects the decontaminated tooth from future infections.1 The removal of microorganisms from the root canal is an important step for the success of endodontic therapy.2 Because of the high complexity of the transversal anatomy of the root-canal system including anatomic irregularities, mechanical instrumentation alone cannot sufficiently remove the smear layer (a layer of dentine debris mixed with organic components created during the shaping process that adheres to the canal walls and blocks the dentinal tubules) from dentinal surfaces, which may lead to the deeper penetration of bacteria in the dentinal tubules.2,3 Furthermore, mechanical instrumentation is not capable of completely eradicating the microbial contaminations.2,3 For an increase in the effectiveness of disinfection and the acquisition of a bacteria-free root-canal space, antimicrobial irrigation solutions are used during endodontic treatment. The most frequently utilized endodontic irrigation solution contains sodium hypochlorite (NaOCl) with a concentration in the range 0.5−5.25% (w/w), which has bactericidal activity and the © 2017 American Chemical Society

Received: September 13, 2017 Accepted: September 18, 2017 Published: September 18, 2017 34762

DOI: 10.1021/acsami.7b13929 ACS Appl. Mater. Interfaces 2017, 9, 34762−34772

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ACS Applied Materials & Interfaces

introduced in a 250 mL round-bottom flask, and kept at 95 °C under reflux for 1 h. Once the temperature was equilibrated, 50 mg of AgNO3 was added to the reaction medium and stirred for 5 min. A 2 ml portion of 3 wt % aqueous sodium citrate solution, which was preheated to 95 °C, was then added. A progressive color change appeared within minutes. The reaction was kept at 95 °C for 1 h, which resulted in a milky yellow-gray suspension of AgNPs. This suspension was then cooled down, followed by centrifugation and washing with UPW several times (3 × 100 mL, 5000 rpm). The final solution was purged with Argon and stored in the dark at 4 °C. 2.2. Synthesis of AgNPs@SiO2. Prior to the encapsulation of AgNPs with porous silica shell, the first step was the preparation of cetyltrimethylammonium nitrate (CTAN), which was obtained from CTA−bromide using an anion exchange resin (Amberlite IRA-40). First, a ∼15 cm × 3 cm (height × diameter) ion-exchange column was set up by using approximately 30 g of resin material. The resin was washed with saturated KNO3 until all bound Cl− counterions of the resin material were exchanged with NO3−. After the column was washed with UPW, drops of AgNO3 solution (approximately 0.5 mol/ L) were added into the collected water, to see if there is any color change due to precipitation of AgCl. No color change indicates that all bound chlorides are successfully removed. A 5 g portion of CTAN was dissolved in 100 mL of water with the help of ∼5 mL of ethanol, which was eluted through the column. The elution was collected, and water was evaporated under a rotary vacuum evaporator. The concentrate was then dissolved in ethanol, and drops of aqueous AgNO3 solution (approximately 0.5 mol/L) were added to get rid of residual bromides. This step caused most AgNO3 and residual bromides to precipitate as AgBr, which could be removed by filtration. After the filtration step on activated carbon, the solvent was removed under a rotary vacuum evaporator to yield CTAN molecules as white crystals. Synthesized AgNPs were encapsulated with a porous silica shell via the previously reported methods with a few modifications.13,24 The following protocol was used for preparation of 100 mL of AgNP solution. A 500 mg portion of CTAN was dissolved in 3 mL of ethanol and then slowly added to 100 mL of AgNP solution. CTAN was added to the reaction to stabilize the particles and to ensure the porosity of the silica layer on the surface of AgNPs. The reaction was stirred for 1 h to ensure the homogeneous distribution of CTAN in the solution. Once the CTAN had been fully dispersed, 200 μL of tetraethylorthosilicate (TEOS) and 50 μL of 3-aminopropyltriethoxysilane (APTES) were added, separately. After 24 h, the solution was centrifuged for 15 min at 5000 rpm to precipitate the AgNPs@SiO2, and the supernatant was removed. After this step, the particles were washed with ethanol (10 × 40 mL, 5000 rpm), and the solution was sonicated for 10 min between each cleaning cycle. Subsequently, AgNPs@SiO2 in 100 mL of ethanol were stirred overnight to ensure the porous characteristic of the silica shell since CTAN was highly soluble in ethanol. As a final step, the particles were centrifuged for 30 min at 5000 rpm to obtain the particle pellets, which were kept under vacuum for 3 h to evaporate residual ethanol. A 100 mL portion of UPW was then added to disperse AgNPs@SiO2. Synthesized particles were stored in the dark at 4 °C until further use. 2.3. Preparation of Irrigation Solutions. The synthesized AgNPs and AgNPs@SiO2 were digested in HNO3 to determine the Ag content of the particles. The inductively coupled plasma mass spectroscopy (ICP−MS) measurements revealed the actual Ag concentration of the synthesized stock solutions of AgNPs and AgNPs@SiO2 as 2.40 and 2.16 mM, respectively. Upon dilution of these silver-containing solutions, series of irrigation solutions, which consist of 0.18 mM Ag, were prepared to be used in our further experiments. Two different irrigation solutions were formulated in this study: 0.18 mM AgNPs@SiO2 + 0.75 mM Tris + 3% (w/w) NaOCl + 35% (w/w) SP; and 0.18 mM AgNPs@SiO2 + 0.75 mM Tris + 3% (w/w) NaOCl + 35% (w/w) EGTA in UPW at pH 7.5. Tris(hydroxymethyl)aminomethane (Tris) was added to prevent the precipitation of silver chloride (AgCl) since NaOCl can act as a chloride supplier. Solutions were first prepared without NaOCl, and their pH values were adjusted with NaOH in such a way that the pH

interest due to their broad-spectrum antibacterial activities.9−14 They have been widely exploited in the biomedical industry for the development of novel bacteria-resistant products such as catheters, surgical coatings, wound dressings, medical implants, and dental materials.15−19 However, real-world applications of silver-containing nanomaterials are often hindered by the ease of oxidation or photoreduction of these nanoparticles under ambient conditions, which leads to aggregation and significant reduction in their antimicrobial activity.12,13,20 As a solution to this problem, various polymers have been deployed to encapsulate and stabilize AgNPs.11,14,21,22 Although such polymers can efficaciously improve the stability and the antibacterial performance of AgNPs, they often necessitate complex and tedious synthetic pathways with high cost. Encapsulation of the core nanoparticles with a silica shell improves colloidal stability with relatively easy regulation of the encapsulation process.13,23,24 In addition, silicon-based materials are usually regarded as highly biocompatible. Surprisingly, Ag@Si (core−shell) particles have been rarely employed for antimicrobial applications although the shell can play a significant role in protecting the Ag core. Compared to the conventional silica surface, the porous silica layer offers the advantage of being able to slowly release the inorganic core materials. Silver ions released from the oxidized surface of Ag nanoparticles are believed to be the main active species that inhibits growth of bacteria, although the exact action of the mechanism is not yet fully understood.14,20,25 Therefore, porous silica material shows huge potential to be used in the encapsulation of the Ag core since it provides channels for silver ions to pass through. Given our interest to improve the outcomes of root-canal treatment, we fabricated porous SiO2-coated AgNPs (AgNPs@ SiO2), which can be used in the development of irrigation solutions of the next generation to drastically improve the longterm antimicrobial effect of the irrigant and to avoid reinfection of the root canals. To ease and shorten the treatment, we developed all-in-one irrigation solutions, which can be prepared just prior to the treatment by simply mixing chelating agents [e.g., sodium phytate (SP) or ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA)] with our choice of disinfectant (e.g., Ag nanoparticles). It was shown for the first time that prevention of single- and multispecies biofilm regrowth can be achieved for at least 7 days by using AgNPs@SiO2 in combination with suitable cleaning compounds. In addition, ex vivo studies with real tooth samples demonstrated that the irrigation solutions can effectively remove the smear layer, contributing to further disinfection of the root canals. For evaluation of the potential of the proposed solutions for future applications, cytotoxicity tests were performed toward human gingival fibroblasts, revealing lower toxicity of our irrigation solutions compared to the conventionally used one (3% NaOCl and 17% EDTA). This proof-of-concept study demonstrates the possibilities for obtaining irrigation solutions for endodontic or root-canal treatment with long-term antimicrobial activities by using AgNPs@SiO2.

2. EXPERIMENTAL SECTION All chemicals and solvents were purchased from Sigma-Aldrich and used without further purification unless otherwise stated. 2.1. Synthesis of Citrate-Protected AgNPs. For the synthesis of AgNPs, a modified version of the conventional citrate reduction method was used.26 A 100 mL portion of ultra pure water (UPW) was 34763


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gauge side-vented needle was filled with the irrigation solution containing AgNPs@SiO2. The root canal was irrigated after each file change and as a final rinse with the same solution for 1 min. Total irrigation time did not exceed 5 min. For biofilm development, teeth were prepared as described by Zhang et al.29 Briefly, teeth were immersed with 3% NaOCl for 1 min, and then 10 mL of 17% EDTA was added for 2 min to remove the smear layer. Teeth were washed three times with deionized water (dH2O) and dried afterward. Next, the samples were steam-autoclaved for 20 min under 15 psi pressure at 121 °C to ensure that no bacteria remained and stored at room temperature for biofilm formation.29 2.6. Establishment of Single- and Multispecies Biofilms. Fusobacterium nucleatum ATCC 10953, Actinomyces naeslundii ATCC 12104, and Enterococcus faecalis ATCC 29212 were obtained from American Type Culture Collection (Manassas, VA). Streptococcus sanguinis DSM 20068 was obtained from The Leibniz Institute DSMZGerman Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany), and Streptococcus sobrinus OMZ 176 was kindly provided by the Center of Dental Medicine, University of Basel (UZM). For single-species biofilm development, sterile white nontransparent polystyrene (PS) 96-well plates (Brand, Wertheim, Germany), hydroxyapatite (HA) discs (Clarkson Chromatography Products Inc., South Williamsport, PA; steam-autoclaved for 20 min under 15 psi pressure at 121 °C), and the pre-prepared teeth described above were coated with diluted horse serum (DHS; 10% in 0.9% NaCl) for 2 h at 37 °C. E. faecalis preculture was centrifuged at 11 300g for 5 min (MiniSpin Plus, Eppendorf AG, Hamburg, Germany), and pellets were washed with 1 mL of 0.9% NaCl twice. Finally, the pellet was resuspended with 0.9% NaCl to an optical density (OD600) of 1.00 ± 0.05. A 16-fold dilution of bacteria suspension in fluid universal medium (FUM) with 10% horse serum was added to the test surfaces and incubated anaerobically for 10 days at 37 °C. Medium was changed every other day. After 4 days, new fresh medium was added and left for 3 more days. The protocol for the establishment of the five-species biofilm was adapted from Brandle et al.30 Briefly, for precultures, F. nucleatum and A. naeslundii were cultivated in thioglycolate (Biomerieux, Marcyl’Étoile, France) supplemented with menadione and hemin, for 3 days at 37 °C with shaking (40 rpm) under anaerobic conditions. In parallel, after 2 days incubation of F. nucleatum and A. naeslundii, S. sanguinis and S. sobrinus were inoculated and grown in thioglycolate supplemented with menadione, hemin, and E. faecalis in Schaedler bouillon at 37 °C with shaking (40 rpm) under anaerobic conditions for 1 day. Similar to that of the E. faecalis single-species biofilm formation, all precultures with a culture volume of 5 mL each were centrifuged at 11 300g for 5 min (MiniSpin Plus, Eppendorf AG, Hamburg, Germany), and pellets were washed with 1 mL of 0.9% NaCl twice. Finally, the pellet was resuspended with 0.9% NaCl to an OD600 of 1.00 ± 0.05. A 16-fold dilution of each bacteria suspension in FUM with 10% horse serum was mixed, added to the test surfaces, and incubated anaerobically for 10 days at 37 °C. Medium was changed every other day. After 4 days, new fresh medium was added and left for 3 more days. 2.7. Irrigation Tests with Biofilms. After biofilm development, media were removed, and biofilms were washed with dH2O to remove unattached cells. Biofilms were then treated with irrigation solutions for 5 min at room temperature. Untreated biofilms were used as control. Irrigation solutions were removed after treatment, and biofilms were washed three times with dH2O. Either biofilms were directly quantified, or fresh Schaedler medium was added for further incubation of 48, 96, and 168 h to assess the long-term antibacterial activity. 2.8. Preparation of Irrigated Teeth Samples for SEM Analysis. Before analysis of the teeth surfaces with SEM, samples were fixed by applying Kanovsky fixing solution [4% paraformaldehyde, 2.5% glutaraldehyde, 1 × PBS (phosphate-buffered saline)] for 1 h at room temperature. Afterward, samples were carefully washed three times with 1 × PBS and dehydrated by ethanol (30 min in 50% ethanol; 30 min in 70% ethanol; 30 min in 80% ethanol; 60 min in

will be at 7.5 when they are combined with NaOCl. NaOCl was added to the solutions just prior to conducting the experiments. 2.4. Materials Characterization. Transmission electron microscopy (TEM) photographs were obtained with a Spirit BioTWIN instrument to observe the morphology and the porosity of the synthesized AgNPs@SiO2. Acceleration voltage for TEM was 80 kV. In the sample preparation, particles were diluted in ethanol and cast onto a copper grid. The size distributions were analyzed using a threshold-based particle analysis in the ImageJ program. For analysis of the dissolution of the Ag core of AgNPs@SiO2, 3% NaOCl was added to the solutions of 0.18 mM AgNPs@SiO2 + 0.75 mM Tris + 35% EGTA or SP. After 5 min at room temperature the mixture was centrifuged at 5000 rpm for 15 min. The pellets were washed with UPW several times (5 × 30 mL) via centrifugation and then dispersed in ethanol and drop-cast onto a copper grid for TEM observations. Scanning electron microscopy (SEM) observations were performed on an FEI XLF30-FEG instrument, under an acceleration voltage of 1.50 kV. Samples were mounted on aluminum stubs using carbon double-face tape on the surface prior to SEM observation. X-ray photoelectron spectroscopy (XPS) measurements were carried out using a PHI VersaProbe II scanning XPS microprobe (Physical Instruments AG). Analysis was performed using a monochromatic Al Kα X-ray source of 24.8 W power with a beam size of 100 μm. The spherical capacitor analyzer was set at 45° takeoff angle with respect to the sample surface. The pass energy was 46.95 eV yielding a full width at half-maximum of 0.91 eV for the Ag 3d5/2 peak. Curve fitting was performed using PHI Multipak software. UV−vis spectroscopy measurements were performed on a PerkinElmer Lambda 25 UV−vis spectrometer. Measurements with ICP−MS (Thermo Scientific) were performed to analyze the silver in suspensions. A 1 mL portion of Schaedler broth medium was added to each irrigation solution, and the medium was changed every other day as follows: Solutions were centrifuged at 12 000 rpm for 5 min, and the pellet was collected and resuspended in the fresh medium. After 4 days, new fresh medium was added and left for 3 more days. The final supernatant was collected after centrifugation, filtered through syringe filters with 0.2 μM pore size, and used for ICP−MS analysis. The average ζ-potential and the hydrodynamic particle size distribution were measured by a Malvern Zetasizer Nano ZS instrument equipped with a maximum 4 mW He−Ne laser, emitting at 633 nm. Measurements were performed using Malvern disposable polycarbonate folded capillary cells with gold-plated beryllium−copper electrodes (DTS1070), which were rinsed with ethanol and ultrapure water before filling. The temperature was set to 25 °C, and the viscosity values used were those of pure water. Results are reported as the arithmetic mean and related standard deviation of the peaks’ means. 2.5. Preparation of the Tooth Samples for Smear-Layer Removal and Biofilm Assays. Single-root human adult teeth (noncarious) (Dentsply Maillefer, Ballaigues, Switzerland) with closed apexes were extracted. Radiographs were used to find the single root and determine the root-canal shape (round or oval). The goal was to have the same population of teeth for each irrigation solution. The teeth were stored at 4 °C in 0.9% sodium chloride supplemented with 0.02% sodium azide to prevent bacterial growth. The clinical crown was removed to standardize the root length at 15 mm using a diamond bur. First, a manual 10 K file (COLORINOX, Dentsply Maillefer) was inserted beyond the apex to confirm patency (1 mm subtracted to establish the working length). The canal and glide path were enlarged with a PATHFILE (P1 and P2, Denstply Maillefer) system. The teeth were then shaped with the crown-down technique to size X3 (PROTAPER NEXT, Dentsply Maillefer). Slightly different shaping techniques can be found in the literature due to variations of the size of teeth used in different studies.27,28 For application of the conventional irrigation technique, a 30 gauge side-vented needle (PRORINSE, Dentsply Maillefer) was filled with 3% NaOCl solution and used between each file passage. A solution of 17% EDTA (CanalPro, Coltene) was used as a final rinse for 1 min. For the application of new formulations developed in this study, a 30 34764


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Figure 1. Morphological analysis of the synthesized Ag nanoparticles. (A) TEM image of AgNPs. Inset shows UV−vis absorption spectrum of AgNPs in water. (B−D) TEM images of AgNPs@SiO2 with different magnifications, of which part D shows the TEM image at higher magnification to observe the porous structure of the silica shell. (E, F) Histograms showing particle size distributions of AgNPs and AgNPs@SiO2, respectively. 90% ethanol; and 60 min in 100% ethanol). Samples were incubated in hexamethyldisilazane (HMDS), which was removed carefully after 30 min, and subsequently, the samples were dried inside a desiccator until gold sputtering (10 nm). Gold sputtering was not applied for the samples used for smear-layer removal. 2.9. Bacterial Viability Assay. For measurement of the bacterial cell viability of the biofilms formed in the microplates, after treatment and washing, 100 μL of Schaedler bouillon was added to each well, and the plates were closed with silver sticking foil (Aluma Seal IITM, Sigma). After vortexing for 10 min, droplets on the cover were spun down at 1000g (centrifuge 5430R, Eppendorf AG, Hamburg, Germany) for a few seconds. Silver foil was removed, and 100 μL of BacTiter-Glo reagent (Promega, Fitchburg, WI) was added to each well. Plates were incubated for 5 min in the dark at room temperature. The luminescence intensity was measured with the Synergy HT MultiDetection microplate reader (BioTek, Luzern, Switzerland; time, 1s; emission filter, empty; gain, 135). Biofilms formed on HA discs were evaluated similarly: HA discs were placed in wells of sterile 12-well plates, 1 mL Schaedler medium was added, and plates were closed with silver sticking foil. After biofilm formation, plates were vortexed vigorously for 10 min, followed by a gentle sonication (20 W, 5 s). A 100 μL portion of bacteria suspension was added to wells of a white 96-well plate (3 repetitions per HA disc), and 75 μL of Luciferase reagent (BactiterGlo) was added on top. Plates were incubated for 5 min in the dark at room temperature, and the viable cells were analyzed as described above. All data were analyzed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA) software. Analysis of the statistical differences between two samples was performed by one-way ANOVA and Tukey−Kramer’s post hoc test. The statistical significance is defined as follows: *P < 0.05, **P < 0.01, ***P < 0.001. 2.10. Cytotoxicity Assay. Irrigation solutions were examined for their cytotoxicity toward human gingival fibroblasts (HGFs, ScienCell, Carlsbad, CA) using ISO10993-5. HGF cells were seeded with 15 000 cells per well (200 μL volume) in a 96-well microtiter plate (TPP

Techno Plastic Products AG, Trasadingen, Switzerland). Cells were incubated overnight at 37 °C with 5% CO2. Hereafter, the HGF cells were treated with different irrigation solutions for 10, 60, and 120 min. Cells without treatment served as control. Cell viability was determined via MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay to determine metabolic activity of the HGF cells [3 repeatitions (wells) of the same treatment]. Yellow MTT is reduced to purple formazan in the mitochondria of living cells. This color change can be directly related to the number of viable (living) cells and was evaluated using the Synergy HT Multi-Detection microplate reader (BioTek, Luzern, Switzerland).

3. RESULTS AND DISCUSSION 3.1. Characterization of the Synthesized Nanoparticles. The prepared nanoparticles were first visualized using TEM, as shown in Figure 1A−D. The bare AgNPs are nearly spherical with an average diameter of 119 ± 29 nm (Figure 1E). The average sizes and standard deviations of the AgNPs were determined by ignoring nanoparticles with triangle, polygon, or rodlike shape. These irregularly shaped particles were found to contribute to less than 5% of the total AgNP population, as suggested by UV−vis data presented in the inset of Figure 1A. The relatively sharp absorption peak of AgNPs at 437 nm wavelength suggests low polydispersity in terms of the shape and the size. The core−shell structure of AgNPs@SiO2 can be clearly distinguished as illustrated in Figure 1C, providing direct verification of effective silica coating. The TEM image presented in Figure 1D was taken at higher magnification to observe porosity of the silica shell. The darker area in the vicinity of the Ag core was only observed when the focus of the beam was fixed to observe the pores clearly. The histogram in 34765


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Figure 2. (A) XPS wide-scan spectrum of AgNPs@SiO2. (B, C) Corresponding high-resolution XPS spectra of N 1s and Ag 3d, respectively. (D) Oxidative dissolution curves of 0.18 mM AgNPs + 0.75 mM Tris, and 0.18 mM AgNPs@SiO2 + 0.75 mM Tris in the presence of 3% NaOCl at pH 7.5. Measurement was conducted at 450 nm.

corresponding to Si 2p, Si 2s, C 1s, Ag 3d5/2, Ag 3d3/2, N 1s, and O 1s electrons are clearly observed (Figure 2A). The broad N 1s peak which was deconvoluted into two spectral bands at 399.18 and 401.11 eV in Figure 2B corresponds to the protonated and the unprotonated form present in APTES. The doublet intense peaks at 367.7 and 373.9 eV as shown in Figure 2C are for the 3d5/2 and 3d3/2 electrons of Ag, which are the characteristic peaks for metallic-state Ag. With the aid of XPS

Figure 1F provides the size distribution of AgNPs@SiO2 as 258 ± 73 nm. UV−vis spectra of AgNPs@SiO2 are not presented here since it is not possible to mark a sharp absorption peak corresponding to AgNPs because of the highly light-scattering medium originating from relatively large sizes of the silica shell. Synthesized particles were characterized further by XPS to investigate the chemical constitution. Figure 2A−C depicts an XPS survey spectrum of AgNPs@SiO2. The characteristic peaks 34766


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Figure 3. Representative SEM images of the middle-third of the root canals before and after irrigation treatment for 5 min: (A) without treatment, (B) precleaned sample, (C) AgNPs@SiO2 + Tris + NaOCl, (D) AgNPs@SiO2 + Tris + NaOCl + EGTA, (E) AgNPs@SiO2 + Tris + NaOCl + SP, and (F) EDTA + NaOCl, conventional and sequential treatment served as a benchmark. The final pHs of irrigation solutions were adjusted to 7.4− 7.6. The optimum pH of an endodontic medicament is chosen as close as possible to that of body fluids (pH 7.4−7.5). NaOCl is mixed with the AgNPs@SiO2-containing irrigation solution just before the treatment. The final concentrations of the different components were adjusted as follows: 3% NaOCl, 17% EDTA, 0.75% Tris, 35% SP, 35% EGTA, 0.18 mM AgNPs, and 0.18 mM AgNPs@SiO2.

year still exhibited excellent antimicrobial activity (data not shown). This suggests that prepared solutions can be stored for a long period without compromising their stability. 3.2. Evaluation of Smear-Layer Removal. In the next step, we sought to find suitable cleaning agents, which can effectively remove the smear layer of teeth samples, with the goal of a one-step treatment. Currently, the standard endodontic procedure involves EDTA as a decalcifying agent and NaOCl as disinfectant and an agent to dissolve organic tissues. However, NaOCl is not effective in the presence of EDTA. Therefore, EDTA has to be applied with NaOCl consecutively. Carboxylic-acid-containing compounds, which show high affinity to calcium, or phosphonic-acid-containing molecules, the oxides of hydroxyapatites on the tooth surface, were investigated as cleaning agents. The selection criteria were that the compounds should effectively remove the smear layer of teeth samples and at the same time be compatible with AgNPs@SiO2 and NaOCl. After extensive screening of 14 molecules (SI, Table S2) with different concentrations, two compounds, sodium phytate (SP) and ethylene glycol-bis(βaminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA) with a final concentration of 35% (w/w), were identified to be able to remove most of the smear layer and leave most dentinal tubules open. Consequently, two active irrigation solutions, which consist of 0.18 mM AgNPs@SiO2, 0.75 mM Tris, and 35% SP or EGTA in dH2O at pH 7.5, were formulated. These two irrigation solutions mixed with 3% (final concentration) NaOCl were first studied for their ability to remove the smear layer of teeth samples. The smear layer may act as a substrate for bacteria, allowing their penetration in the dentinal tubules and limiting the optimum penetration of disinfecting agents.3 Hence, it should be removed to increase the success of the endodontic treatment. Since the performance of irrigation solutions has been observed to be better in the middle-third of the root canal compared to other regions,27,28 we focused our

data, it was shown that the surface of AgNPs was successfully covered with amine-functionalized silica material. For investigation of the release profile of Ag+ species from AgNPs@SiO2 during the course of treatment (typically 60−90 min), oxidative dissolution kinetics of AgNPs@SiO2 was measured by UV−vis spectrophotometry in the presence of our choice of oxidizer, 3% NaOCl (Figure 2D). Tris was also added to prevent the precipitation of silver chloride (AgCl) since NaOCl releases chloride upon reaction. Measurements were conducted at the fixed wavelength of 450 nm, which overlaps with the surface plasmon resonance peak of synthesized bare AgNPs. It is shown in Figure 2D that the release profile of Ag+ species from bare AgNPs is much faster than that of AgNPs@SiO2, which demonstrates the advantage of having the SiO2 shell around AgNPs to ensure the activity of the particles even after the course of treatment. In addition, the sharp increase seen in the profile of bare AgNPs (Figure 2D) can be attributed to the aggregation of AgNPs in the presence of chloride ions (Cl−), increasing the scattered light intensity. This aggregation is not observed for AgNPs@SiO2 because of the protective silica shell, resulting in a smoother decrease. The hydrodynamic radius and the electrostatic stabilization of AgNPs@SiO2 were studied in various solutions after 5 min of mixing the nanoparticle-containing solution with NaOCl by dynamic light scattering and the ζ-potential analyzer. Corresponding values are presented in the Supporting Information (SI; Table S1). The results suggest that the particles mostly remain as small aggregates (a couple of particles together). Therefore, sonication of 2−5 min was needed prior to usage of the particles to obtain a relatively homogeneous dispersion. The stability of particles in the absence of NaOCl was analyzed after repeated intervals (3 and 6 months), and no significant shift in either radius or ζpotential values was observed. Furthermore, the AgNPs@SiO2 stored in the irrigation solutions (without NaOCl) for almost 1 34767


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Figure 4. Irrigation of (A) E. faecalis and (B) multispecies biofilms with the solutions developed in this study. Biofilms were treated with the indicated solutions for 5 min at room temperature. Untreated biofilm served as the control. After irrigation, fresh medium was added to the biofilm, and regrowth of bacteria was evaluated after 48, 96, and 168 h, respectively. The viable cells in the untreated biofilms were set as 100%. Asterisks denote statistical significance as follows: *P < 0.05, **P < 0.01, ***P < 0.001. These were assigned to the corresponding controls at 0, 48, 96, and 168 h, respectively (N = 3). The final concentrations of the different components were adjusted as follows: 3% NaOCl, 17% EDTA, 0.75% Tris, 35% SP, 35% EGTA, 0.18 mM AgNPs, and 0.18 mM AgNPs@SiO2.

were either directly quantified or further incubated with fresh medium for an additional 48, 96, or 168 h to measure the longterm antibacterial activity. We found that treatment with all tested endodontic solutions led to a significant reduction of viable cells in both single- and 5-species biofilms (Figure 4, 0 h). Compared to the untreated control, more than 95% reduction in viable cells was achieved for almost all solutions except AgNPs@SiO2 + Tris + NaOCl + EGTA, which led to 40% and 85% reduction of the singlespecies and 5-species biofilms, respectively (Figure 4, 0 h). It seems that EGTA interacts with Ag species in the presence of NaOCl, which decreases the antimicrobial efficiency of the solution. However, it is difficult to speculate the underlying interactions because of the usage of the complex media. When the biofilms were incubated with fresh media for a further 48, 96, or 168 h, biofilms treated with the conventional irrigation solution containing EDTA + NaOCl or with solutions containing bare AgNPs already allowed biofilm regrowth after 48 h (Figure 4, 48 h). In contrast, the biofilms treated with solutions containing AgNPs@SiO2 hardly showed any regrowth, even after 168 h, except the solution AgNPs@SiO2 + Tris + NaOCl + EGTA which allowed regrowth after 168 h (Figure 4). In the absence of NaOCl, the AgNPs@SiO2containing solutions performed as good as or even better, in the case of AgNPs@SiO2 + Tris + EGTA, than the solutions with NaOCl in preventing biofilm regrowth (Figure 4), demonstrating that the medium contains oxidative components to oxidize AgNPs@SiO2 constantly. These results are in good agreement with the oxidative dissolution data shown in Figure 2D, as the release of Ag+ species from AgNPs is much faster than that from AgNPs@ SiO2. This explains the low antimicrobial effect of AgNPs for the long term in comparison to the AgNPs protected by the

analysis on that part of the canal to check effectiveness of our proposed solutions. Figure 3 shows the representative SEM images of the middle-third region of different tooth samples after ex vivo treatment with the irrigation solutions. Compared with the untreated sample (Figure 3A) and the clean sample (Figure 3B), the treatment with AgNPs@SiO2 + Tris + NaOCl solution, lack of cleaning agent, did not remove the smear layer, and no open dentinal tubes could be seen (Figure 3C). In contrast, both mixtures containing cleaning agent EGTA or SP were found to be highly effective for smear-layer removal and allowed opening of most dentinal tubes (Figure 3D and E), similar to the benchmark of conventional and sequential treatment with EDTA and NaOCl (Figure 3F). These results demonstrate that AgNPs@SiO2 particles cannot play a significant role in smear-layer removal; instead, the binds EGTA and SP are the important factor in such a removal. The aggregates seen on the tooth sample in Figure 3E are likely caused by the detachment of dentine materials from different parts of the teeth into the root canal during the cutting process. 3.3. Irrigation of Single- and Multispecies Biofilms Formed on Microplates. We further investigated the antimicrobial activity of the newly developed irrigation solutions with the aim to demonstrate the long-term effect of AgNPs@SiO2 after treatment. For this, a single-species biofilm of E. faecalis (a typical pathogen found in root-canal infection31) and a 5-species biofilm consisting of known oral pathogens F. nucleatum, A. naeslundii, E. faecalis, S. sanguinis, and S. sobrinus were developed for 10 days in 96-well plates, respectively, under anaerobic conditions. The biofilms were treated with the irrigation mixtures for 5 min at room temperature, mimicking the clinical treatment conditions. Untreated biofilms were used as control. Irrigation solutions were removed, and biofilms were washed with dH2O. Biofilms 34768


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Figure 5. Irrigation of E. faecalis biofilm formed on HA discs. The 10-day-old biofilms were treated with the indicated solutions for 5 min at room temperature. Untreated biofilm served as control. After irrigation, fresh medium was added to the biofilm, and regrowth of bacteria was evaluated after 48, 96, and 168 h, respectively. The viable cells in the untreated biofilms were set as 100%. The final concentrations of the different components were adjusted as follows: 3% NaOCl, 17% EDTA, 0.75% Tris, 35% SP, 35% EGTA, and 0.18 mM AgNPs@SiO2. Asterisks denote statistical significance as follows: *P < 0.05, **P < 0.01, ***P < 0.001. These were assigned to the corresponding controls at 0, 48, 96, and 168 h, respectively (N = 3).

Figure 6. Representative SEM images of root canals of real teeth after formation of E. faecalis biofilm for 10 days and treatment with irrigation solutions. (A) Tooth control surface without any biofilm. (B) E. faecalis biofilm without any treatment. Biofilms were treated for 5 min at room temperature with (C) NaOCl + EDTA, (D) AgNPs@SiO2 + Tris + NaOCl + EGTA, (E) AgNPs@SiO2 + Tris + EGTA, (F) AgNPs@SiO2 + Tris + NaOCl + SP, and (G) AgNPs@SiO2 + Tris + SP. Scale bar: 2 μm. The final concentrations of the different components were adjusted as follows: 3% NaOCl, 17% EDTA, 0.75% Tris, 35% SP, 35% EGTA, and 0.18 mM AgNPs@SiO2.

application of AgNPs@SiO2 resulted in a long-term antimicrobial effect of the irrigation treatment for at least 168 h, whereas conventional treatment with EDTA and NaOCl already led to loss of the activity within 48 h (Figure 5). E. faecalis biofilms on real tooth surfaces were difficult to analyze quantitatively because of undefined surfaces and irregular shapes of different individual samples. SEM imaging was thus applied to qualitatively investigate the irrigation results. For each irrigation treatment, three teeth, each with three images at different locations, were evaluated. Figure 6 shows the representative SEM images. Without any treatment, E. faecalis biofilms cover the root canals in multiple layers so that tubules are almost not visible (Figure 6B). After treatment with the irrigation solutions, the bacteria loads are dramatically reduced, but not completely removed (Figure 6C−G). It seems that solutions containing AgNPs@SiO2 and SP removed

silica shell. Thus, application of AgNPs@SiO2 leads to the desired long-term antimicrobial effect. A comprehensive comparison of different mixtures for their disinfection efficiency is provided in the Supporting Information, Figure S1. Even though treatment with the mixture of AgNPs@SiO2 + Tris + NaOCl also abolished the growth of biofilms (Figure 4), it was not able to remove the smear layer of the tooth samples effectively (Figure 3B); thus, it is not suitable as an irrigation solution for endodontic root treatment. 3.4. Irrigation of Biofilms Formed on HA Discs and Tooth Surfaces. To further study whether the developed irrigation solutions can also effectively irrigate biofilms formed on HA discs and on real tooth samples, 10-day-old biofilms of E. faecalis on top of these surfaces were treated. HA discs were used here as they have well-defined surfaces, and the irrigation efficiency can be evaluated quantitatively. It was found that 34769


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Figure 7. Amount of Ag present in supernatants of AgNPs@SiO2 measured by ICP−MS. Solutions containing AgNPs@SiO2 were incubated in bacterial growth medium at 37 °C, as described in the Experimental Section. The available Ag in the supernatant was measured after 7 days. The final concentrations of the different components were adjusted as follows: 3% NaOCl, 0.75% Tris, 35% SP, 35% EGTA, and 0.18 mM AgNPs@SiO2. N = 3.

Figure 8. Determination of HGF cell viability after exposure to different endodontic irrigation solutions. The viable HGF cells subjected to distilled water were used as control and set as 100%. The cytotoxic cutoff was set to 70% viable cells, i.e., a lethal dose of 30% (LD30). Conventional treatment (NaOCl + EDTA) was set as the benchmark, which signifies a lethal dose of 85% (LD85). The final concentrations of the different components were adjusted as follows: 3% NaOCl, 17% EDTA, 0.75% Tris, 35% SP, 35% EGTA, and 0.18 mM AgNPs@SiO2. N = 3.

Previously, it has been reported that the minimal inhibition concentration of silver is, depending on tested bacteria and growth conditions, between 0.024 and 635 μM.36 The available silver measured here should be enough to be lethal for a wide range of bacteria species, which correlates well with what was obtained in this work (Figures 4 and 5). Observation of Ag+ in the absence of NaOCl is also in good agreement with the results shown in Figures 4 and 5, where the antibacterial activity was detected in the absence of NaOCl even after 168 h, indicating that the medium contains oxidative agents, which can oxidize AgNPs@SiO2 constantly. Our results suggest that, for real applications, in the case of residual Ag@SiO2 particles in the root canal after the treatment, the antimicrobial activity can persist for the long term. We also analyzed the effect of the oxidation process on the silver core in the presence of NaOCl. For this, two irrigation solutions containing all of the components were prepared for TEM analysis. After the treatment for 5 min, ∼60% of the core AgNPs were found to dissolve in the oxidized environment on the basis of the TEM images, resulting in empty core−shell particles (SI, Figure S2). Solutions containing either EGTA or SP showed a similar ratio of etched core particles compared to total number of nanoparticles. 3.6. Cytotoxicity. Even though silver and silver-based compounds are well-known to be antimicrobial against a broad

biofilm most effectively (Figure 6F,G), which is in agreement with the results obtained from treatment of biofilms on microplates (Figure 4), where SP showed almost complete inhibition of biofilm regrowth after 168 h. 3.5. Dissolution of Ag from AgNPs@SiO2. The excellent long-term antimicrobial activity of AgNPs@SiO2 demonstrated above prompted us to further investigate the correlation of released Ag with the activity. Although the antimicrobial effect of AgNPs has been widely described, their mechanism of action is yet to be completely understood.20,32−34 Despite the many conflicting opinions in the literature regarding the mode of action, recent observations discovered that it is likely that the underlying antimicrobial mechanism is largely propelled by the oxidative dissolution of the nanoparticles to generate Ag+ ions.20,25,35 In our system, we speculated that monovalent silver species (Ag+) produced from oxidative dissolution of the AgNPs@SiO2 act as an antibacterial agent, while the nanoparticles act as a reservoir, allowing the slow release of Ag+. Here, we quantitatively measured the amount of available Ag from AgNPs@SiO2 after long-term incubation of nanoparticles in bacterial growth medium. It was found that, after 168 h (7 days), a sufficient amount of Ag was still detected in AgNPs@ SiO2 supernatant, with 418 μM in AgNPs@SiO2 + Tris + NaOCl + SP solution as the highest and 63 μM in AgNPs@ SiO2 + Tris + NaOCl + EGTA as the lowest (Figure 7). 34770


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ACS Applied Materials & Interfaces spectrum of bacteria, their cytotoxicity is of great concern.12 It has been reported that AgNPs are biocompatible with mouse fibroblast and human osteoblast cells37 and have exhibited minimal perturbation to human cells.37−40 Here, we examined the cytotoxicity of the different endodontic solutions toward human gingival fibroblasts (HGFs) using protocol ISO10993-5 (Figure 8). The viable HGF cells exposed to distilled water were used as control and set as 100%. The cytotoxic cutoff is set at less than 70% viable cells of the control samples [lethal dose of 30 (LD30)]. The conventionally used irrigation solution containing 3% NaOCl and 17% EDTA (sequentially applied) was used as a benchmark (LD85). We observed that the benchmark exhibited a high cytotoxicity with about 15% cell viability of the control samples. The two newly developed irrigation solutions EGTA + AgNPs@SiO2 + Tris + NaOCl and SP + AgNPs@SiO2 + Tris + NaOCl, showed about 25% and 40% cell viability of the control samples, respectively. Thus, the new solutions have lower cytotoxicity than the widely used solution, even after prolonged contact of up to 2 h (Figure 8).

E.E., B.G., F.Z., S.A., and B.L.O. performed the experiments and analyzed the data. Q.R., E.E., and B.G. drafted and wrote the manuscript. All authors read and approved the final manuscript. Funding

This study was funded by the Swiss federal Commission for Technology and Innovation (CTI) Grant 13945.2 PFLS-LS. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Nikolaos Nianias for helping with XPS experiments.

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4. CONCLUSIONS This proof-of-concept study for the first time demonstrates the long-term antimicrobial potential of the nanoparticle-based approach for endodontic infection treatment. Prevention of biofilm regrowth can be achieved by using AgNPs@SiO2 in combination of suitable cleaning compounds, which do not lose activity when applied as an all-in-one solution. The AgNPs@ SiO2-containing solutions are proven less cytotoxic than the classically used ones. This study forms the groundwork for an infection treatment process where the smear layer of dentinal surfaces of instrumented root canals can be removed, and bacterial regrowth after treatment can be limited for at least 7 days. Meanwhile, in contrast to the current treatment method where NaOCl and EDTA are used sequentially, the developed solutions allow one-step handling by the simple mixing of two pre-irrigation solutions (NaOCl, and AgNPs@SiO2 + Tris + EGTA or SP) right before the treatment, which hence decreases the treatment time and is beneficial to both patients and dentists. This nanoparticle-based approach may be applicable for proactive long-term disinfection management on biomedical devices such as urinary catheters and endoscopes.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b13929. ζ-potential values, hydrodynamic radii, and list of 14 molecules that were screened as cleaning agents; additional figures of the irrigation of biofilms and dissolution of Ag from AgNPs@SiO2 (PDF)

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ABBREVIATIONS SP, sodium phytate EGTA, ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′tetraacetic acid EDTA, ethylenediaminetetraacetic acid CTAN, cetyltrimethylammonium nitrate TEOS, tetraethylorthosilicate APTES, aminopropyltriethoxysilane ICP−MS, inductively coupled plasma mass spectroscopy Tris, tris(hydroxymethyl)aminomethane TEM, transmission electron microscopy SEM, scanning electron microscopy XPS, X-ray photoelectron spectroscopy UV−vis spectroscopy, ultraviolet−visible spectroscopy HA, hydroxyapatite DHS, diluted horse serum FUM, fluid universal medium PBS, phosphate-buffered saline HGF, human gingival fibroblast MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide dH2O, deionized water

AUTHOR INFORMATION

Corresponding Authors

*E-mail: Francesco.Stellacci@epfl.ch. *E-mail: [email protected]. ORCID

Francesco Stellacci: 0000-0003-4635-6080 Author Contributions ⊥

E.E. and B.G. equally contributed to this paper. E.E., B.G., S.M., F.S., and Q.R. conceived and designed the experiments. 34771


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Core-shell silver nanoparticles in endodontic disinfection solutions

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