Sonochemical Coatings of ZnO and CuO Nanoparticles Inhibit

Jul 25, 2012 - Free ZnO and CuO NPs inhibited biofilm formation of Streptococcus mutans. Furthermore, by using the sonochemical procedure, we were abl...
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Sonochemical Coatings of ZnO and CuO Nanoparticles Inhibit Streptococcus mutans Biofilm Formation on Teeth Model Michal Eshed,† Jonathan Lellouche,†,‡ Shlomo Matalon,§ Aharon Gedanken,*,† and Ehud Banin*,‡ †

Kanbar Laboratory for Nanomaterials, Department of Chemistry, The Bar-Ilan Institute of Nanotechnology and Advanced Materials, and ‡The Biofilm Research Laboratory, The Bar-Ilan Institute of Nanotechnology and Advanced Materials, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel § Department of Oral Rehabilitation, School of Dental Medicine, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel ABSTRACT: Antibiotic resistance has prompted the search for new agents that can inhibit bacterial growth. We recently reported on the antibiofilm activities of nanosized ZnO and CuO nanoparticles (NPs) synthesized by using sonochemical irradiation. In this study, we examined the antibacterial activity of ZnO and CuO NPs in a powder form and also examined the antibiofilm behavior of teeth surfaces that were coated with ZnO and CuO NPs using sonochemistry. Free ZnO and CuO NPs inhibited biofilm formation of Streptococcus mutans. Furthermore, by using the sonochemical procedure, we were able to coat teeth surfaces that inhibited bacterial colonization.

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INTRODUCTION Oral biofilms, also called plaque, are complex three-dimensional structures consisting of diverse and multispecies bacterial communities. More than 500 known species have been isolated from the mouth.1 Development of oral biofilms begins with the formation of a conditioning saliva-derived film on the tooth surface followed by attachment of primary colonizers to hostderived receptor molecules present in the acquired pellicle. These early colonizers than promote subsequent interactions with secondary colonizers that join the growing biofilm creating a mature multispecies microbial community.2 Oral biofilms are known to contribute to the development of oral diseases such as gingivitis and dental caries, which are among the most common infectious diseases and are of major public health concern.3 Oral biofilm can form on enamel, implants, or orthodontic devices. An initial step in dental plaque formation is the adherence of oral bacteria to the acquired pellicle that coats the tooth’s enamel surface. This process involves several forces, including hydrophobic and ionic bonds, as well as lectin-like interactions between bacterial adhesions and complementary receptors on the host surface.4 Oral Streptococci have been shown to be the major primary colonizers of clean enamel surfaces, and these organisms constitute 60−80% of dental plaque.5 Moreover, Streptococcus mutans (S. mutans) plays an important role in the development of dental caries6 due to the microbial production of acids that subsequently dissolve tooth enamel.7 Unfortunately, the ability to eradicate the dental biofilm is extremely difficult, and simply brushing the teeth is not sufficient. Thus, approaches that will inhibit or delay oral biofilm formation can greatly improve oral health. Metals such as silver, gold, zinc,8,9 and metal oxide10,11 nanoparticles (NPs) have been © 2012 American Chemical Society

shown to have strong antimicrobial activity against a broad range of pathogenic microorganisms. However, the effects of ZnO and CuO on the formation of oral biofilms have not been widely reported. The objective of the current study was to present a new method, via ultrasound irradiation, for coating the surface of artificial teeth with ZnO/CuO NPs, and to characterize whether these coated surfaces can restrict bacterial colonization and biofilm formation. Sonochemical irradiation has been proven as an effective technique for the synthesis of nanophased materials, as well as for the deposition and insertion of NPs on/into mesoporous ceramic and polymer supports, fabrics, and glass.12,13 In sonochemical synthesis, stable NP coatings are formed by the high energy created by the collapse of the cavitation bubbles. This collapse creates very high temperatures and pressure, conditions leading to the rupture of chemical bonds. According to the interpretation suggested for the sonochemical coating process, microjets are formed after the collapse of the acoustic bubble near a solid surface. These microjets throw the newly formed NPs at the solid substrate at such a high speed (>200 m/s) that the NPs can penetrate the surface. This Article examines the use of a chemical-sonication process to generate ZnO and CuO NPs and their subsequent deposition on the surface of artificial acryl tooth before implantation. We also examined the antibiofilm properties of NP/hydroxyapatite pellets (HA),14 a major component and an essential ingredient of normal teeth. The antibiobilm activity of these NPs and coated surfaces (i.e., Received: April 8, 2012 Revised: July 3, 2012 Published: July 25, 2012 12288

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1 h in titanic acid and a glutamate solution in 4:5 ratio concentrations, respectively. After three cycles of washing with PBS, the samples were exposed to an osmium tetraoxide solution for 1 h. Finally, water residue was removed with water−ethanol and ethanol−freon solutions (from 50% to 100% of each solvent). Samples were then dried in air, chromium-coated, and imaged by HR-SEM (JEOL-6700F, accelerating voltage 15 kV) apparatus. Biofilm Assays on a HA/NP Mixture. Pellets were prepared as follows: 100 mg of the HA powder was mixed and pressed with 2 mg of ZnO or CuO NP powder (2% wt). We evaluated the antibiofilm activity of the mixture using two biofilm models (static and continuous culture flow): (i) For the static model, the pellets were placed in a 24-well plate (Greiner Bio-One). Each well contained a 3 mL bacterial suspension of S. mutans at a final concentration of an OD595 = 0.15 (approximately 1.5 × 108 CFU/mL) solution in BH media. After incubation for 24 h at 37 °C, the pellets were washed twice with ddH2O and stained using the Live/Dead BacLight kit (Molecular Probes, Invitrogen) according to the manufacturer’s protocol. Viable bacteria with intact cell membranes are stained in green (SYTO9), whereas dead bacteria with damaged membranes are stained in red (propidium iodide). Both the excitation/emission maxima for these two dyes are 480/500 nm for the SYTO9 stain and 490/635 nm for the propidium iodide one. Direct observation of the biofilms formation was by confocal laser scanning microscopy (CLSM, Leica SPE). Obtained images were further processed by the Imaris image analysis software (Imaris v.6.0, Bitplane Scientific Software). (ii) A flow cell system was inoculated with a 0.3 OD595 dilution of S. mutans bacteria overnight culture. The flow was initiated after 1 h with a flow rate of 10 mL per hour. The system was incubated at 37 °C for 24 h, stained, and imaged using the same Live/Dead staining. Reactive Oxygen Species Measurements. ESR Measurements. 5,5-Dimethyl-pyrroline N-oxide (DMPO) (0.02 mol/L) was added to 1 mg/mL of a CuO or ZnO NP aqueous suspension and was drawn by a syringe into a gas-permeable Teflon capillary (Zeus Industries, Raritan, NJ), 0.032 inner diameter, 0.015 wall thickness, and 15 cm length. Each capillary was folded twice, inserted into a narrow quartz tube that was open at both ends, and then placed in the ESR cavity. The ESR spectra were recorded on a Bruker ER 100d X-band spectrometer, before or after adding 100 mL of the cell culture (1.5 × 108 CFU/mL). The measurements were repeated at least four times for each sample. The microwave of the ESR was set at a frequency of 9.67 GHz and the power at 20 mW. Measurement conditions were as follows: scan width, 65 G; resolution, 1024; receiver gain, 2 × 105; conversion time, 81.92 ms; time constant, 655.36 ms; number of scans, 2. Calibration of the ESR Spectrometer. For conversion of the ESR spin adduct signal to concentrations, the experimental double integral signal of DMPO−OH was compared to the double integral of a stablefree radical spectrum of known concentrations of 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) measured with identical settings.

teeth and HA) against S. mutans is presented. Our results suggest a promising strategy for antibiofilm activity that can be further developed to improve oral health.

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METHODS

ZnO and CuO NPs Synthesis and Coating Procedure. 0.02 g of zinc acetate [Zn(O 2 CCH 3 ) 2 ·2H 2 O] or copper acetate [Cu2(OAc)4·H2O] (purchased from Aldrich and used without further purification) was dissolved in 10 mL of double-distilled water (ddH2O). To this solution was added ethanol for a final volume of 100 mL with an ethanol/water ratio of 10:1. To achieve a basic pH, NH3·H2O was added until the pH value of the solution was ∼8. The reaction mixture was irradiated for 30 min with a high-intensity ultrasonic horn (Ti horn, 20 kHz, 750 W at 60% efficiency). The sonication vessel was placed in a cooling bath, maintaining a constant temperature of 30 °C during the reaction. The obtained solution was centrifuged, and the resulting precipitating products were washed twice with ddH2O and then with ethanol, after which the obtained NPs were dried under vacuum. NP coatings were obtained by placing an artificial acryl tooth (teeth were obtained from the School of Dental Medicine at the Tel Aviv University) directly into the sonochemical reaction medium according to the methodology described above. The tooth was held by a wire to keep it at a constant distance of 2 cm from the sonicator’s tip during the entire reaction process. The amount of ZnO and CuO NPs deposited during the synthesis was determined indirectly by inductively coupled plasma (ICP, ULTIMA 2, Horiba Scientific). Teeth that were coated by ZnO and CuO NPs were first washed three times with ddH2O and then immersed in a strong acid (HNO3), so the zinc and cupric ion concentrations in the solution were determined. To analyze the coating morphologies, the samples were coated with chromium and imaged by HR-SEM (JEOL-6700F, accelerating voltage 15 kV). The size distribution of the NPs was determined from the measurement of images taken with HR-SEM (n = 100). To evaluate the coating distribution on the surface, we mapped the oxygen, zinc, and copper elemental distributions on the surfaces by energy X-ray dispersive spectroscopy (EDS) (EDAX apparatus on FEI-Inspect S). Elemental mapping was performed at both 15 keV and 0.58 nA with a resolution of 133 eV. Maps were created in most cases from 100 scan frames using a dwell time of 100 μs and a 512 × 384 pixel/frame resolution. Bacterial Cultures and Growth Conditions. In all of the experiments, S. mutans 700610 (clinical isolate) was grown aerobically at 37 °C in a brain heart (BH) medium supplemented with 0.5% sucrose (noted BH), which is known to induce robust biofilm formation.15 Antibacterial Test. Overnight cultures of tested bacteria were diluted (1:100) in fresh media and grown for 8 h at 37 °C (shaking, 250 rpm). Water-insoluble compounds were assayed in a modified macrodilution assay. ZnO and CuO NP samples at various concentrations (0.0001−1.0 mg/mL) were added to sterile polypropylene tubes (Greiner Bio-One), and the appropriate volume of a 0.01 OD595 solution (approximately 107 CFU/mL) of S. mutans in media was added. 100 μL of the tested cell suspension was then added to each well of a 96-well plate that was incubated for 24 h at 37 °C. Following incubation, bacterial growth was determined spectrophotometrically by measuring the absorbance at 595 nm (OD595, Synergy 2, BioTek Instruments). Biofilm Assays on Artificial Tooth. Artificial teeth were assayed using a static biofilm assay.16 Teeth were placed in a 24-well plate (Greiner Bio-One). Each well contained a 3 mL bacterial suspension of S. mutans at a final concentration of OD595 = 0.15 (approximately 1.5 × 108 CFU/mL) in BH media. After 24 h incubation at 37 °C, the teeth were washed twice with ddH2O to remove the nonattached cells, and the biofilm biomass was stained with 1% crystal-violet (CV, Sigma) for 15 min at room temperature. The stained biofilm, which was formed on the teeth, was washed five times with ddH2O, and the remaining CV was eluted by the addition of absolute ethanol for 15 min. The biofilm biomass was then determined by measuring the absorbance at OD595. To examine the biofilm morphology, teeth samples were exposed after incubation to Karnovsky’s fixative (glutaraldehyde + paraformaldehyde) for 1 h. The samples were washed three times with a phosphate buffer saline (PBS) without Ca2+ and Mg2+. Samples were immersed for

Figure 1. Effect of ZnO and CuO NPs on S. mutans growth. Growth curves of S. mutans exposed to ZnO (1 mg/mL) and CuO NPs (1 mg/ mL) for 24 h at 37 °C. Untreated bacteria served as a negative control. Error bars represent the standard deviation of three independent experiments conducted in triplicate. 12289

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Figure 2. Imaging of sonochemical ZnO and CuO NP teeth coating. Teeth were coated using a sonochemical procedure described in the experimental section. HR-SEM images in two different magnifications of tooth surfaces coated with (A,B) ZnO NPs and (D,E) CuO NPs are presented. (C) Size distribution of ZnO and (F) CuO NPs present on the teeth surfaces. (G,H) The surface of uncoated teeth.

Figure 3. Distribution of the ZnO and CuO NPs coating on teeth surfaces. (A) Illustration of a tooth with two arrows that reveal two different areas of mapping analysis. (B) Elemental mapping analysis of the two teeth coated with ZnO and CuO NPs, respectively. Intracellular ROS Assays. For the detection of reactive oxygen species (ROS) production upon exposure to CuO and ZnO NPs, the cells were preincubated with 10 μM of CM-H2DCFDA [5-(and 6)chloromethyl-2,7-

dichlorodihydrofluorescein diacetate, acetyl ester] (Invitrogen, Molecular Probes) in PBS for 30 min, allowing the dye to enter the cells. This dye freely permeates the cell membrane and becomes fluorescent upon cleavage by 12290

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ROS. Cells were washed twice with PBS before fluorescent microscopy (Confocal Microscope, ZEISS). Samples were analyzed at an excitation of 485 nm and an emission at 535 nm. Untreated and CM-H2DCFDA preincubated cells with 100 μM H2O2 for 30 min were used as negative and positive controls, respectively.

Pseudocatalase Assay. The detection of pseudocatalase activity was measured by using the Amplex Red reagent-based assay as follows: cells were preincubated with an Amplex Red (Invitrogen, Molecular Probes) reagent solution and horseradish peroxidase (HRP), an HRP (Invitrogen, Molecular Probes) stock solution for 30 min at 37 °C. In the assay, pseudocatalase first reacts with H2O2 to produce water and oxygen, after which the Amplex Red reagent reacts with a 1:1 stoichiometry with any unreacted H2O2 molecule in the presence of HRP to produce the highly fluorescent oxidation product, resorufin. Therefore, as psedo-catalase activity increases, the signal from resorufin decreases. Superoxide Dismutase (SOD) Assay. Cells were incubated with ZnO and CuO NPs for 4 h at 37 °C. The suspensions were then centrifuged for 10 min. In the next step, a solution of trichloroacetic acid (TCA, SigmaAldrich) in 10 °C was added to each sample to cause cell lysis. In addition, nitro blue tetrazolium (NBT, Invitrogen), NBT, was added, and thus its concentration in the sample was 0.45 mM. All samples were then incubated at 37 °C for an additional 20 min. First, the superoxide ions (O2−) convert NBT to NBT-diformazan, and then the absorbance at 450 nm was read by using a microplate reader (Synergy 2, BioTek Instruments).

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RESULTS AND DISCUSSION Antimicrobial Activity of ZnO and CuO NPs. We first examined the activity of ZnO and CuO NPs in suspension on S. mutans growth. Bacterial cultures of S. mutans were inoculated with ZnO and CuO NPs, and the growth was monitored over a period of 24 h (Figure 1). Increasing the concentrations of both NPs, from 0.0001 to 1.0 mg/mL, did not result in the growth inhibition of S. mutans. Even when the highest concentration of both NPs (1.0 mg/mL) was tested, the growth of S. mutans was not affected. Previous studies have suggested that ZnO and CuO NPs inhibit S. mutans growth.17,18 One possible explanation for this difference is in the strains tested and/or the growth conditions. The previous studies grew the bacteria in anaerobic conditions, while our test was in aerobic conditions. We tested the activity of our ZnO NPs also under anaerobic conditions, and the results were similar, that is, antibiofilm activity without growth inhibition (data not shown). Thus, differences are most likely attributed to differences in the strains used in each study. Characterization and Antibiofilm Properties of ZnO and CuO NP-Coated Surfaces. S. mutans is well-known for its ability to form biofilms in the oral niche.19 Thus, our next step was to examine the ability of the NPs to block biofilm formation. Teeth were coated with either ZnO or CuO NPs using sonochemistry. It is important to note that coating teeth is challenging due to their complex surface topography and hardness. The deposition of ZnO and CuO NPs was characterized by HR-SEM (Figure 2). The sonication reaction

Figure 4. ZnO and CuO NP-coated teeth restrict S. mutans biofilm formation. Coated and uncoated teeth were incubated with S. mutans for 24 h. (A) The biofilm biomass that developed was stained by a crystal-violet staining as described in the experimental section. (B) Quantification of biofilm biomass. (C) Planktonic growth. Error bars represent the standard deviation of three independent experiments conducted in triplicate.

Figure 5. Antibiofilm properties of ZnO and CuO NP-coatings. HR-SEM imaging of S. mutans biofilms on coated and uncoated teeth. Biofilms were grown for 24 h at 37 °C. 12291

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Figure 6. Antibiofilm properties of ZnO NPs/HA and CuO NPs/HA pellets. CLSM images of S. mutans biofilm grown in static and flow cell models on ZnO or CuO NPs/HA pellets after 24 h at 37 °C. HA pellet used as a negative control. Green and red staining represent live and dead bacterial cells, respectively. Images obtained by CLSM represent the general trend seen in three independent experiments.

biofilm biomass was quantified. From the results presented in Figure 4, it is clear that the ZnO and CuO NP-coated teeth reduced biofilm formation by 85% and 70%, respectively (Figure 4B), as compared to the uncoated tooth surface that supported massive biofilm formation (Figure 4A). Careful examination of the tooth topography displays the natural preference of the biofilm formation to be developed on teeth slits (Figure 4A). These results were also supported by HR-SEM imaging (Figure 5). No S. mutans biofilm formation was observed on NP-coated teeth as compared to the uncoated control tooth in which dense colonization was observed (Figure 5). Another interesting aspect is the external planktonic growth observed in the media surrounding the teeth (Figure 4C). Growth was not affected, and the values measured were similar to those obtained with the uncoated teeth. This corresponds to the results obtained with the free NPs, in planktonic cultures, and further confirm that these NPs have no antimicrobial activity against S. mutans planktonic cultures. This conclusion fits well with the growth curves of S. mutans (Figure 1) exposed to even higher concentrations of free ZnO or CuO NPs (1 mg/mL). Biofilm Assays on HA/NPs Mixture. To further characterize the antibiofilm properties, we incorporated the ZnO and CuO NPs into HA. Antibiofilm activity was examined using two biofilm models, static and continuous flow-cell system. Figure 6 demonstrates CLSM images of S. mutans biofilm formation on NPs/HA pellets after 24 h using the static- (Figure 6A) and continuous-flow cell (Figure 6B) models. In both models, the pellets were challenged with fresh bacterial cultures for one day. As can be seen in Figure 6, HA pellets that contain NPs are able to completely inhibit S. mutans biofilm formation, whereas HA pellets without NPs supported massive biofilm formation, and many of the cells within the biofilm were viable (i.e., the green cells). Moreover, by the comparison of the two control pellets (under static and dynamic conditions), biovolume [μm3] of the green cells was 7784 and 5719, respectively, while in the case of the red cells the biovolume [μm3] was calculated as 10 419 and 8226, respectively.

Figure 7. Reactive oxygen species measurements by ESR. ESR spectra for ZnO and CuO NPs in the presence of S. mutans suspension and with the spin trap DMPO.

created a uniform coating over the entire tooth. The size distribution of ZnO NPs was between 120 and 180 nm, whereas for CuO NPs the size was 18−20 nm (Figure 2C,F). Figure 2G and H shows the surface of uncoated teeth. The topography of the tooth poses a major challenge for coating due to its nonplanar surface that contains dents and protrusions. To examine the coating, elemental mapping analysis was carried out on the top of the tooth and on the wall side of the tooth. The results are presented in Figure 3. The two arrows on the illustrated tooth in Figure 3A mark the two different areas that were mapped. Figure 3B presents the spread of Zn, Cu, and O elements across the tooth’s surface. This analysis suggests that the sonication coating process did achieve a homogeneous coating across the entire tooth surface. The concentrations of zinc oxide and cupric oxide were 0.075 and 0.1 g/cm2, respectively. Following the coating characterization, the ZnO and CuO NPcoated teeth were tested for their ability to restrict S. mutans attachment and subsequent biofilm development. The teeth were challenged for 24 h in the presence of S. mutans, and the 12292

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Figure 8. Intracellular ROS detection assay. No intracellular ROS generation was detected in the control sample, and was scarcely noticed when adding ZnO and CuO NPs. H2O2 served as a positive control. The red and blue markers appear as solid circles in the images and represent two areas in the sample, the bacteria cell and the background, and in the graphs, these are represented by arrows that point to the intensity of the green fluorescence, which belong to the two markers.

In previous studies, our group examined the mechanism by which ZnO NPs exert antibacterial activity. One mechanism is through the generation of radical oxygen species (ROS), mainly, hydroxyl radicals, that cause cellular damage (e.g., membrane and DNA).20 To further characterize this phenomena, 5,5-dimethyl1-pyrroline-N-oxide (DMPO) was used for trapping hydroxyl radicals followed by ESR measurements of S. mutans cell

suspension exposed to ZnO or CuO Nps. The results presented in Figure 7 clearly indicate that ROS were generated in a solution of S. mutans cells exposed to either ZnO or CuO NPs. Surprisingly, despite the fact that ZnO and CuO NPs are known to release ROS,20,21 the generation of ROS neither harmed nor affected S. mutans growth. According to work done in our lab, ZnO and CuO NPs demonstrated an excellent 12293

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bactericidal effect against Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) cultures.22,23 In other words, although there is clear evidence of ROS generation, these ROS do not kill S. mutans or inhibit its growth. However, it could potentially prevent biofilm formation on the surface. Our hypothesis is that during oxidative stress, proteins involved in adhesion and biofilm formation may be damaged. Furthermore, the expression of different genes, which are responsible for attachment of bacteria to the surface and biofilm formation, could be inhibited. In fact, it was previously suggested that oxidative stress appears to minimize Streptococcal biofilms.24 Next, we tested whether we can detect ROS inside the cells upon exposure to ZnO and CuO NPs and compared this to the suspension of S. mutans without NPs. The intracellular ROS were measured using a fluorescent dye (CM-CH2DCFDA), as described in the Methods section. As can be seen in Figure 8, exposure to H 2O 2 (our positive control) yielded high fluorescence, while only low fluorescence was measured in the samples containing ZnO or CuO NPs. This most likely indicates that there is a lower presence of ROS inside the cells. As we can see from Figure 8, in all four images, the intensity of the green fluorescence of the background (represented by the blue marker) was zero. The NP treated samples showed a slightly higher fluorescence as compared to the background. However, the positive control treated sample (H2O2) was 10-fold higher. Taken together, these results suggest the cells exposed to the NPs only accumulate a small amount of oxidative radicals. The fact that the intracellular ROS level in S. mutans is low may suggest the bacteria can induce antioxidant activity to protect itself. To examine this possibility, we tested the activity of major oxidative stress enzymes (superoxide dismutate (SOD) and pseudocatalase). As can be seen in Figure 9, SOD and

showed that CuO and ZnO NPs are uniformly deposited onto the surface of a tooth with sizes of 30 and 150 nm, respectively. The ZnO and CuO NP-coated teeth show significant reduction in biofilm formation by 85% and 70%, respectively, as compared to uncoated tooth, which supported massive biofilm formation. Our preliminary results suggest that both types of NPs generate ROS in the solution, and this triggers the bacteria to induce SOD and pseudocatalase enzymes to cope with the ROS. This most likely protects S. mutans and explains why growth inhibition was not observed upon exposure to the NPs. The mechanism by which the NPs cause biofilm inhibition still remains unknown and will require further study. Still, this activity warrants further characterization of ZnO and CuO NPs as potential antibiofilm coatings for dental implants.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (A.G.), [email protected]. il (E.B.). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS NP/NPs, nanoparticle/nanoparticles; ZnO, zinc oxide; CuO, copper oxide; S. mutans, Streptococcus mutans; HA, hydroxyapatite; ddH2O, double-distilled water; ICP, inductively coupled plasma; HR-SEM, high-resolution scanning electron microscope; EDS, energy X-ray dispersive spectroscopy; BH, brain heart; OD, optical density; CFU, colony forming unit; CV, crystal violet; PBS, phosphate buffer saline; CLSM, confocal laser scanning microscope; ESR, electron spin resonance; DMPO, 5,5dimethyl-pyrroline N-oxide; TEMPO, 2,2,6,6-tetramethylpiperidine-N-oxyl; ROS, reactive oxygen species; CM-H2DCFDA, 5-(and 6)chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester; HRP, horseradish peroxidase; NBT, nitro blue tetrazolium; SOD, superoxide dismutase; CCR2, CC chemokine receptor 2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine receptor 5; TLC, thin layer chromatography

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REFERENCES

(1) Moorem, W. E.; Moore, L. V. The Bacteria of Periodontal Disease. Periodontol 2000 1996, 5, 66−77. (2) Leung, K. P.; Crowe, T. D.; Abercrombie, J. J.; Molina, C. M.; Bradshaw, C. J.; Jensen, C. L.; Luo, Q.; Thompson, G. A. Control of Oral Biofilm Formation by an Antimicrobial Decapeptide. J. Dent. Res. 2005, 84, 1172−1177. (3) Selwitz, R. H.; Ismail, A. I.; Pitts, N. B. Dental Caries. Lancet 2007, 369, 51−59. (4) Murray, P. A.; Prakobphol, A.; Lee, T.; Hoover, C. I.; Fisher, S. J. Adherence of Oral Streptococci to Salivary Glycoproteins. Infect. Immun. 1992, 60, 31−38. (5) Diaz, P. I.; Chalmers, N. I.; Rickard, A. H.; Kong, C.; Milburn, C. L.; Palmer, R. J., Jr.; Kolenbrander, P. E. Molecular Characterization of Subject-Specific Oral Microflora During Initial Colonization of Enamel. Appl. Environ. Microbiol. 2006, 72, 2837−2848. (6) Hamada, S.; Slade, H. D. Biology, Immunology, and Cariogenicity of Streptococcus mutans. Microbiol. Rev. 1980, 44, 331−384. (7) Leverett, D. H. Fluorides and the Changing Prevalence of Dental Caries. Science 1982, 217, 26−30. (8) Phan, T. N.; Buckner, T.; Sheng, J.; Baldeck, J. D.; Marquis, R. E. Physiologic Actions of Zinc Related to Inhibition of Acid and Alkali Production by Oral Streptococci in Suspensions and Biofilms. Oral Microbiol. Immunol. 2004, 19, 31−38. (9) Hernandez-Sierra, J. F.; Ruiz, F.; Cruz Pena, D. C.; MartinezGutierrez, F.; Martinez, A. E.; De Jesus Pozos Guillen, A.; Tapia-Pérez, H.;

Figure 9. Measuring SOD and pseudocatalase activities in S. mutans. Samples containing NPs were compared to the control sample. The activities of both enzymes are higher during exposure to the NPs than in the control sample. Error bars represent the standard deviation of three independent experiments conducted in triplicate.

pseudocatalase activities in S. mutans are slightly increased upon exposure to the NPs as compared to the control. This result may explain the increased resistance of the planktonic cultures to the oxidative stress. Future work will be required to further elucidate the mechanism underlying the antibiofilm activity associated with ZnO and CuO NPs.

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CONCLUSIONS The present research describes teeth coating using the sonochemistry method. SEM analysis on the teeth surfaces 12294

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dx.doi.org/10.1021/la301432a | Langmuir 2012, 28, 12288−12295