Nanocatalysts promote Streptococcus mutans biofilm matrix degradation and enhance bacterial killing to suppress dental caries in vivo

Abstract

Dental biofilms (known as plaque) are notoriously difficult to remove or treat because the bacteria can be enmeshed in a protective extracellular matrix. It can also create highly acidic microenvironments that cause acid-dissolution of enamel-apatite on teeth, leading to the onset of dental caries. Current antimicrobial agents are incapable of disrupting the matrix and thereby fail to efficiently kill the microbes within plaque-biofilms. Here, we report a novel strategy to control plaque-biofilms using catalytic nanoparticles (CAT-NP) with peroxidase-like activity that trigger extracellular matrix degradation and cause bacterial death within acidic niches of caries-causing biofilm. CAT-NP containing biocompatible Fe3O4 were developed to catalyze H2O2 to generate free-radicals in situ that simultaneously degrade the biofilm matrix and rapidly kill the embedded bacteria with exceptional efficacy (>5-log reduction of cell-viability). Moreover, it displays an additional property of reducing apatite demineralization in acidic conditions. Using 1-min topical daily treatments akin to a clinical situation, we demonstrate that CAT-NP in combination with H2O2 effectively suppress the onset and severity of dental caries while sparing normal tissues in vivo. Our results reveal the potential to exploit nanocatalysts with enzyme-like activity as a potent alternative approach for treatment of a prevalent biofilm-associated oral disease.

Keywords: Antibacterial; Biofilms; Catalysis; Dental caries; Extracellular matrix; Iron oxide; Nanoparticles.

Copyright © 2016 Elsevier Ltd. All rights reserved.

Figures

Figure 1. CAT-NP retention and spatial distribution within 3D biofilm structure

a , Scanning electron microscopy (SEM) of the morphology of untreated biofilm and (b) treated with CAT-NP (CAT-NP bound; see arrows). Magnified view of CAT-NP in the selected area (b1). SEM/EDS images showing iron (pink) distribution on biofilms (b2). c, CAT-NP bound on biofilm as determined by measuring iron amounts with ICP-MS. d, 3D architecture of untreated biofilm. e, Spatial distribution of CAT-NP in treated biofilm: f, CAT-NP (white); g, bacteria (green); h, EPS (red) are observed with confocal microscopy. i, Cross-sectional merged images of top and middle areas of the biofilm. j, Orthogonal distribution of CAT-NP, and (k) bacteria and EPS across biofilm thickness

Figure 2. CAT-NP activity within biofilms with pH dependent catalysis in situ

a, Catalytic activity of CAT-NP adsorbed within biofilms. Inset: photographic images of CAT-NP treated biofilm before and after exposure to H2O2 and TMB (the blue color indicates free-radical generation via H2O2 catalysis in situ). b, Catalytic activity of CAT-NP (0.5 mg ml-1) treated biofilms at different pH. The difference of absorbance values between 2a and 2b is due to different incubation time used for each experiment. For figure 2a, we conducted the incubation with H2O2 and TMB for 30 min to allow all samples generate signals, particularly for those treated with low CAT-NP concentration. For Figure 2b (biofilms treated with 0.5 mg ml-1 CAT-NP), the activity was determined using the standard 5 min incubation with H2O2 and TMB.

Figure 3. Bacterial killing, EPS degradation and biofilm disruption by the combination of CAT-NP and H2O2

a , Viability of S. mutans within CAT-NP treated-biofilms 5 min after H2O2 exposure. b, EPS degradation within biofilm 30 min after H2O2 exposure. c, Degradation of insoluble glucans produced by GtfB and soluble glucans from GtfD. Data are shown as mean ± s.d. *P< 0.001 (vs. control).

Figure 4. Dynamics of biofilm disruption after topical treatments with CAT-NP+H2O2

a Confocal microscopy images at different time points. Biofilms received topical treatment by CAT-NP followed immediately by H2O2 exposure (CAT-NP+H2O2) or sodium acetate buffer (CAT-NP alone) twice daily. For H2O2, biofilms were treated with sodium acetate buffer followed immediately by H2O2 exposure. The control group consisted of biofilms treated with buffer only. Bacterial cells were stained with SYTO 9 (in green) and EPS were labeled with Alexa Fluor 647 (in red). b. COMSTAT analysis of total, cell and EPS biovolume for biofilm at 43h. Data are shown as mean ± s.d. *P < 0.001 (vs. control).

Figure 5. Protection against development of carious lesions by CAT-NP/H2O2 treatment

a , Images of teeth from rats treated as noted. Green arrows indicate initial lesion formation where areas of the enamel is demineralized and become white; blue arrows show moderate carious lesions where areas of enamel are white-opaque or damaged. In some areas, the enamel is eroded leading to cavitation, which are the most severe carious lesions (red arrows). Caries scores are recorded as stages and extent of carious lesion severity according to Larson's modification of Keyes' scoring system: b, Initial lesion (surface enamel white); c, moderate lesion (enamel white-opaque) and extensive (cavitation with enamel eroded and underlying dentin exposed). Data are shown as mean ± s.d. *P ≤ 0.001 (vs. control); ** P ≤ 0.05 (vs. control); δ indicates non-detected.

Figure 6. CAT-NP reduces sHA acid-dissolution

a, Amount of iron released from CAT-NP after incubation at pH 4 or pH 7 via colorimetric assay (Iron Assay Kit, Sigma-Aldrich). b, Amount of remaining sHA after acid-dissolution with or without CAT-NP. c, Optical microscopy (OM) and scanning electron microscopy (SEM) imaging of untreated sHA beads (80 μm diameter), sHA beads in acidic buffer (pH4.5), and sHA beads with CAT-NP in acidic buffer. The data are depicted as mean±s.d.

Figure 7

Schematics of biofilm disruption under acidic conditions by CAT-NP/H2O2in situ.