CoreâShell 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 Bekdemir, Beatrice Gutt, Flavia Zuber, Sergio Allegri, Benjamin Le Ouay, Selma Mefti, Kitty Formentin, Francesco Stellacci, and Qun Ren ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13929 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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ACS Applied Materials & Interfaces

Core-shell silver nanoparticles in endodontic disinfection solutions enable long-term antimicrobial effect on oral biofilms Elif Ertema,#, Beatrice Guttb,#, Flavia Zuberb, Sergio Allegria, Benjamin Le Ouayc, Selma Meftid, Kitty Formentind, Francesco Stellaccia,*, Qun Renb,* a

Institute of Materials, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne CH 1015,

Switzerland b

Laboratory for Biointerfaces, Empa, Swiss Federal Laboratories for Materials Science and

Technology, St. Gallen, Switzerland c

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering,

Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan d

Denstply Sirona, Ballaigues CH 1338, Switzerland

#

Authors equally contributed to this manuscript

*Corresponding authors: [email protected], [email protected]

Keywords: root canal treatment, core-shell silver nanoparticles, irrigation solutions, oral biofilm, multi-species biofilm, antimicrobial activity

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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(β-aminoethyl 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 multi-species biofilms demonstrated for the first time that AgNPs@SiO2 based 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 towards 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 needed long-term antimicrobial effect.

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 Due to 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 shaping process that adheres to the canal walls and blocks the dentinal tubules) from dentinal surfaces, which may


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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 To increase the effectiveness of disinfection and obtain 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 a range of 0.5% and 5.25%, which has bactericidal activity and ability to dissolve organic tissues.4 However, NaOCl is not effective on removal of inorganic components from

the smear

layer,

thus

a

solution

including

a decalcifying

agent

such

as

ethylenediaminetetraacetic acid (EDTA), citric or phosphoric acid should be used.5 This treatment is performed sequentially since the interactions between the two solutions cause loss of NaOCl activity.5 Studies have shown that currently used irrigation media are only moderately effective and 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 sequential treatment are highly desired. The potential benefits of nanotechnology in biomedical field have become widely accepted for the generation of promising strategies to treat various bacterial diseases.8 Silver (Ag) containing nanomaterials are of extensive interest owing to their broad-spectrum antibacterial activities.9-14 They have been widely exploited in biomedical industry for the development of novel bacteriaresistant 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 oxidization or photo-reduction of these nanoparticles under ambient conditions, which leads to aggregation and significant reduction in their antimicrobial activity.1213, 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


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antibacterial performance of AgNPs, they often necessitate complex and tedious synthetic pathways with high cost. Encapsulation of the core nanoparticles with silica shell improves colloidal stability with relatively easy regulation of encapsulation process.13,

23-24

In addition,

silicon based materials are usually regarded as highly biocompatible. Surprisingly, Ag@Si (coreshell) particles have been rarely employed for antimicrobial applications although the shell can play a significant role in protecting the Ag core. Compared to conventional silica surface, 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 mechanism is not yet fully understood.14, 20, 25 Therefore, porous silica material shows huge potential to be used in the encapsulation of 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 in order to drastically improve the long-term 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 further disinfection of the root canals. To evaluate the potential of the proposed solutions for future applications, cytotoxicity tests were performed towards human gingival fibroblasts, revealing lower toxicity of our irrigations solutions compared to the conventionally used one (3% NaOCl and 17% EDTA). This


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proof of concept study demonstrates the possibilities to obtain 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 is stated.

2.1 Synthesis of citrate protected AgNPs To synthesize AgNPs, a modified version of conventional citrate reduction method was used.26 100 ml deionized water (dH2O) was introduced in a 250 ml round-bottom flask, and kept at 95˚C under reflux for 1 hour. Once the temperature was equilibrated, 50 mg of AgNO3 was added to the reaction medium and stirred for 5 minutes. 2 ml of 3 w% 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 hour, and resulted in a milky yellow-grey suspension of AgNPs. This suspension was then cooled down, followed by centrifugation and washing with dH2O several times (3 * 100 ml, 5000 rpm). 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 cetyl trimethylammonium nitrate (CTAN), which was obtained from CTA-Bromide using an anion exchange resin (Amberlite IRA-40). First, ~15 cm * 3 cm (height * diameter) ion-exchange column was set up by using approximately 30 g resin material. The resin was washed with saturated KNO3 until all bound Cl- counter-ion of resin material was exchanged with NO3-. After


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washing the column with dH2O, 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 all bound chlorides are successfully removed. 5 gram of CTAN was dissolved in 100 ml of water with the help of ~5 ml of ethanol and eluted through the column. The elution was collected and water was evaporated under 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 filtration step on activated carbon, the solvent was removed under rotary vacuum evaporator to yield CTAN molecules as white crystals. Synthesized AgNPs were encapsulated with 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 AgNPs solution. 500 mg of CTAN was dissolved in 3 ml of ethanol and then slowly added to 100 ml of AgNPs 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 hour in order to ensure the homogenous distribution of CTAN in the solution. Once the CTAN had been fully dispersed, 200 µl of tetraethylorthosilicate (TEOS) and 50 µl of 3aminopropyltriethoxysilane (APTES) were added, respectively. After 24 hours, 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 minutes between each cleaning cycle. Subsequently, AgNPs@SiO2 in 100 ml ethonal were stirred overnight to ensure the porous characteristic of the silica shell since CTAN was highly soluble in ethonal. 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 hours to


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evaporate residual ethanol. 100 ml of dH2O was then added to disperse AgNPs@SiO2. Synthesized particles were stored in dark at 4 °C until further use.

2.3 Preparation of irrigation solutions The synthesized AgNPs and AgNPs@SiO2 were digested in HNO3 in order 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 mM and 2.16 mM, respectively. Upon dilution of these silvercontaining solutions, series of irrigation solutions, which comprise 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 dH2O 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 were adjusted with NaOH in such a way that the pH 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 Photographs of transmission electron microscopy (TEM) were obtained with Spirit BioTWIN in order 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 program “ImageJ”. To analyze dissolution of 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


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minutes at room temperature the mixture was centrifuged at 5000 rpm for 15 minutes. The pellets were washed with dH2O several times (5 * 30 mL) via centrifugation and then dispersed in ethanol and drop-cast on copper grid for TEM observations. Scanning electron microscopy (SEM) observations were performed on a FEI XLF30-FEG, 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 analyser was set at 45° take-off 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 3d 5/2 peak. Curve fitting was performed using the PHI Multipak software. UV-VIS spectroscopy measurements were performed on a Perkin Elmer Lambda 25 UV-VIS spectrometer. Measurements with ICP-MS (Thermo ScientificTM) were performed to analyze silver in suspensions. 1 ml of Schaedler broth medium was added to each irrigation solutions and the medium was changed every other day as followings: Solutions were centrifuged at 12000 rpm for 5 minutes and the pellet was collected and re-suspended 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 and 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 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


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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 teeth samples for smear layer removal and biofilm assays Single root human adult teeth (non-carious) (Dentsply Maillefer, Ballaigues, Switzerland) with closed apices were extracted. Radiographs were used to find 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% of sodium azide to prevent bacterial growth. The clinical crown was removed to standardize the root length at 15 mm using a diamond bur. First 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 was enlarged with PATHFILE (P1 and P2, Denstply Maillefer). The teeth were then shaped with crown down technique to size X3 (PROTAPER NEXTTM, 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 conventional irrigation technique, a 30 gauge side-vented needle (PRORINSE™, Dentsply Maillefer) was filled with 3% NaOCl solution and used between each file passage. 17% EDTA (CanalPro, Coltene) was used as a final rinse for 1 minute. To apply new formulations developed in this study, a 30 gauge side-vented needle was filled with the irrigation solution containing AgNPs@SiO2. The root canal was irrigated after each file change and as final rinse with the same solution for 1 minute. Total irrigation time did not exceed 5 minutes.


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For biofilm development, teeth were prepared as described by Zhang et al.29 Briefly, teeth were immersed with 3% NaOCl for 1 minute, then 10 ml 17% EDTA was added for 2 min to remove the smear layer. Teeth were washed three times with dH2O and dried afterwards. 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 multi-species biofilms Fusobacterium nucleatum ATCC 10953, Actinomyces naeslundii ATCC 12104 and Enterococcus faecalis ATCC29212 were obtained from American Type Culture Collection (Manassas, USA). Streptococcus sanguinis DSM 20068 was obtained from The Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany) and Streptococcus sorbrinus OMZ 176 was kindly provided by the Center of Dental Medicine, University of Basel (UZM). For single species biofilm development sterile white non-transparent polystyrene (PS) 96-well plates (Brand, Wertheim, Germany), hydroxyapatite (HA) discs (Clarkson Chromatography Products Inc, South Williamsport, USA; steam autoclaved for 20 minutes under 15 psi pressure at 121 °C) and the pre-prepared teeth described above were coated with diluted horse serum (DHS) (1/10 in 0.9 % NaCl) for 2 hours at 37°C. E. faecalis preculture was centrifuged at 11300 g (MiniSpin® plus, Eppendorf AG, Hamburg, Germany) and pellets were washed with 1 ml 0.9 % NaCl twice. Finally the pellet was re-suspended 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 four days, new fresh medium was added and left for three more days.


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The protocol for the establishment of the five species biofilm was adapted from Brändle et al.30 Briefly, for pre-cultures, F. nucleatum and A. naeslundii were cultivated in thioglycolate (Biomerieux, Marcy-l’Étoile, France) supplemented with menadion and hemin, for three days at 37°C with shaking (40 rpm) under anaerobic conditions. In parallel, after two days incubation of F. nucleatum and A. naeslundii, S. sanguinis and S. sorbrinus were inoculated and grown in thioglycolate supplemented with menadion and hemin and E. faecalis in Schaedler Bouillon at 37°C with shaking (40 rpm) under anaerobic conditions for one day. Similar to the E. faecalis single species biofilm formation, all pre-cultures with a culture volume of 5 mL each were centrifuged at 11300 g (MiniSpin® plus, Eppendorf AG, Hamburg, Germany) and pellets were washed with 1 ml 0.9 % NaCl twice. Finally the pellet was re-suspended with 0.9 % NaCl to OD600 of 1.00 +/- 0.05. A 16 fold dilution of each bacteria suspension in FUM with 10% horse serum was mixed and added to the test surfaces and incubated anaerobically for 10 days at 37°C. Medium was changed every other day. After four days, new fresh medium was added and left for three 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 minutes at room temperature. Untreated biofilms were used as control. Irrigation solutions were removed after treatment and biofilms were washed three times with dH2O. Biofilms were either directly quantified or fresh Schaedler medium was added for incubation of further 48, 96 and 168 hours to assess the long term antibacterial activity.

2.8 Preparation of irrigated teeth samples for SEM Analysis


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Before analysis of the tooth surfaces with SEM, samples were fixed by applying Kanovsky-fixing solution (4% Paraformaldehyde, 2.5% Glutaraldehyde, 1 x PBS (Phosphate-buffered saline)) for 1 hour at room temperature. Afterwards, samples were carefully washed three times with 1 x PBS and dehydrated by ethanol (30 minutes in 50% ethanol; 30 minutes in 70% ethanol; 30 minutes in 80% ethanol; 60 minutes in 90% ethanol; 60 minutes in 100% ethanol. Samples were incubated in hexamethyldisilizane (HMDS), which was removed carefully after 30 minutes and subsequently the samples were dried inside a desiccator until gold sputtering (10 nm). Gold sputtering were not applied for the samples used for smear layer removal.

2.9 Bacterial viability assay To measure the bacterial cell viability of the biofilms formed in the microplates, after treatment and washing 100 µl Schaedler Bouillon was added to each well and the plates were closed with silver sticking foil (Aluma Seal IITM, Sigma). After vortexing for 10 minutes droplets on the cover were spun down at 1000 g (Centrifuge 5430R, Eppendorf AG, Hamburg, Germany) for a few seconds. Silver foil was removed and 100 µl BacTiter-Glo™ reagent (Promega, Fitchburg, USA) was added to each well. Plates were incubated for 5 minutes in the dark at room temperature. The luminescence intensity was measured with the Synergy HT Multi-Detection 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 minutes, followed by a gentle sonication (20 W, 5 seconds). 100 µl of bacteria suspension was added to wells of a white 96 well plate (3 repetitions per HA disc) and 75µl Luciferase reagent (BactiterGlo) was added on top. Plates were incubated for 5 minutes in the dark at room temperature, and the viable cells were analyzed as described above.


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All data were analyzed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, USA). 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

CoreâShell Silver Nanoparticles in Endodontic Disinfection Solutions

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CoreâShell Silver Nanoparticles in Endodontic Disinfection Solutions