Simultaneous Photodynamic Eradication of Tooth Biofilm and Tooth Whitening with an Aggregation-Induced Emission Luminogen

Abstract

Dental caries is among the most prevalent dental diseases globally, which arises from the formation of microbial biofilm on teeth. Besides, tooth whitening represents one of the fastest-growing areas of cosmetic dentistry. It will thus be great if tooth biofilm eradication can be combined with tooth whitening. Herein, a highly efficient photodynamic dental therapy strategy is reported for tooth biofilm eradication and tooth discoloration by employing a photosensitizer (DTTPB) with aggregation-induced emission characteristics. DTTPB can efficiently inactivate S. mutans, and inhibit biofilm formation by suppressing the expression of genes associated with extracellular polymeric substance synthesis, bacterial adhesion, and superoxide reduction. Its inhibition performance can be further enhanced through combined treatment with chlorhexidine. Besides, DTTPB exhibits an excellent tooth-discoloration effect on both colored saliva-coated hydroxyapatite and clinical teeth, with short treatment time (less than 1 h), better tooth-whitening performance than 30% hydrogen peroxide, and almost no damage to the teeth. DTTPB also demonstrates excellent biocompatibility with neglectable hemolysis effect on mouse red blood cells and almost no killing effect on mammalian cells, which enables its potential applications for simultaneous tooth biofilm eradication and tooth whitening in clinical dentistry.

Keywords: Streptococcus mutans; aggregation-induced emission; biofilm; photodynamic therapy; tooth whitening.

Figure 1

Photodynamic antibacterial effect of DTTPB. a) Molecular structure of DTTPB. b) Schematic illustration of the simultaneous photodynamic eradication of tooth biofilm and tooth whitening processes with DTTPB. c) Evaluation of the viable state of S. mutans by using a live & dead viability/cytotoxicity assay kit (US Everbright INC.). S. mutans cells were pretreated without/with 10 µm of DTTPB, followed by storage in dark or white‐light irradiation (36 mW cm⁻²) for 10 min. Afterwards, live & dead viability/cytotoxicity assay kit was employed to determine the viable state of S. mutans. 488 nm laser and 515–550 nm emission filter were used for the green channel, while 561 nm laser and 570–620 nm emission filter were used for the red channel. d) S. mutans survival rate evaluated by serial dilution test on BHI agar. S. mutans were treated without/with varied concentrations of DTTPB, followed by storage in dark or white‐light irradiation (36 mW cm⁻²) for 10 min. Data are presented as mean ± standard deviation with at least three replications. e) Representative images of BHI agar plates employed for quantification of S. mutans viability. Both groups were treated with 10 µm of DTTPB. f) Morphology study of S. mutans cells. S. mutans were incubated with designated concentrations of DTTPB, followed by storage in dark or irradiating with white light (36 mW cm⁻²) for 10 min.

Figure 2

Photodynamic inhibition of biofilm formation by DTTPB. a) Images of biofilms formed on culture dish by S. mutans after treatment without/with DTTPB and light irradiation (36 mW cm⁻²) for 10 min. b) Confocal images of the biofilms formed by S. mutans. S. mutans were treated without/with DTTPB and light irradiation (36 mW cm⁻²) for 10 min, cultured for one day, and then stained with NucGreen for 15 min. 488 nm laser and 515–550 nm emission filter were used for S. mutans biofilm imaging. c–e) Photodynamic effect of DTTPB on (c) biofilm biomass, (d) average thickness, and (e) lactic acid production of S. mutans biofilm. Quantification of (c) biofilm biomass, and (d) average thickness of live bacteria were calculated according to 5 random sights of S. mutans biofilms by COMSTAT. For evaluation of lactic acid production, S. mutans were treated without/with 10 µm of DTTPB and light irradiation (36 mW cm⁻²) for 10 min. Data are displayed as mean ± standard deviation. f) Schematic illustration of the biological functions of gtfB, gtfC, gtfD, srtA, nox, and sodA genes. g) Relative RNA expression of gtfB, gtfC, gtfD, srtA, nox, and sodA genes without/with DTTPB and white‐light irradiation (36 mW cm⁻²) treatment. Data are shown as mean ± standard deviation with three replications.

Figure 3

Combination of PDT and CHX for highly effective eradication of S. mutans biofilm in in vitro oral models on glass dishes. a) Delegate 3D images of biofilms treated without/with DTTPB and white‐light irradiation. 1‐day‐old S. mutans biofilms were co‐cultured without/with 20 µm of DTTPB and white‐light irradiation for 10 min (36 mW cm⁻²), treated without/with 0.12% CHX for 30 min, followed by staining with live & dead viability/cytotoxicity assay kit (US Everbright INC.) 488 nm laser and 515–550 nm emission filter were employed the green channel. 561 nm laser and 570–620 nm emission filter were used for the red channel. b) Quantification of survival rate according to the ratio of red/green fluorescence intensity. c,d) Quantification of (c) biofilm biomass, and (d) average thickness of live bacteria according to 5 random sights of S. mutans biofilms by COMSTAT2. Data are displayed as mean ± standard deviation.

Figure 4

a) Schematic illustration of experimental processes for determining the morphology changes of S. mutans biofilms after combined treatment with DTTPB and CHX on glass. b) SEM images of S. mutans biofilms. 1‐day‐old S. mutans biofilms on glass were cultured without/with 20 µm of DTTPB and white‐light irradiation for 10 min (36 mW cm⁻²), and treated without/with 0.12% CHX for 30 min.

Figure 5

Whitening effect of DTTPB. a) Images of coffee‐, tea‐ and cola‐colored sHA treated without/with 20 µm of DTTPB and white‐light (36 mW cm⁻²) irradiation for 10, 30, and 60 min, respectively. b) ΔW of colored sHA treated with 20 µm of DTTPB and white‐light irradiation (36 mW cm⁻²). c) Images of clinical teeth treated with 20 µm of DTTPB without/with white light (36 mW cm⁻²) irradiation or 30% H₂O₂ for 10, 30, and 60 min, respectively and the corresponding SEM images of tooth surfaces after 60 min treatment.

Figure 6

Biocompatibility of DTTPB. a,b) Evaluation of hemolysis induction by DTTPB and 5% H₂O₂. (b) Data are shown as mean ± standard deviation with three replications. c) Evaluation of the toxicity of DTTPB on cells. MRC‐5 cells were incubated with different concentrations of DTTPB or 5% H₂O₂, stained with Calcein AM/PI, followed by imaging with a confocal microscope. The images were merged from green and red channels. 488 nm laser and 500–550 nm emission filter were employed for the green channel. 561 nm laser and 570–620 nm emission filter were used for the red channel.