Oxygen self-sufficient nanodroplet composed of fluorinated polymer for high-efficiently PDT eradicating oral biofilm

Abstract

Oral biofilm is the leading cause of dental caries, which is difficult to completely eradicate because of the complicated biofilm structure. What's more, the hypoxia environment of biofilm and low water-solubility of conventional photosensitizers severely restrict the therapeutic effect of photodynamic therapy (PDT) for biofilm. Although conventional photosensitizers could be loaded in nanocarriers, it has reduced PDT effect because of aggregation-caused quenching (ACQ) phenomenon. In this study, we fabricated an oxygen self-sufficient nanodroplet (PFC/TPA@FNDs), which was composed of fluorinated-polymer (FP), perfluorocarbons (PFC) and an aggregation-induced emission (AIE) photosensitizer (Triphenylamine, TPA), to eradicate oral bacterial biofilm and whiten tooth. Fluorinated-polymer was synthesized by polymerizing (Dimethylamino)ethyl methacrylate, fluorinated monomer and 1-nonanol monomer. The nanodroplets could be protonated and behave strong positive charge under bacterial biofilm acid environment promoting nanodroplets deeply penetrating biofilm. More importantly, the nanodroplets had extremely high PFC and oxygen loading efficacy because of the hydrophobic affinity between fluorinated-polymer and PFC to relieve the hypoxia environment and enhance PDT effect. Additionally, compared with conventional ACQ photosensitizers loaded system, PFC/TPA@FNDs could behave superior PDT effect to ablate oral bacterial biofilm under light irradiation due to the unique AIE effect. In vivo caries animal model proved the nanodroplets could reduce dental caries area without damaging tooth structure. Ex vivo tooth whitening assay also confirmed the nanodroplets had similar tooth whitening ability compared with commercial tooth whitener H₂O₂, while did not disrupt the surface microstructure of tooth. This oxygen self-sufficient nanodroplet provides an alternative visual angle for oral biofilm eradication in biomedicine.

Keywords: AIE photosensitizer; Dental caries; Fluorinated-polymer; Nanodroplets; Oral-biofilm.

Graphical abstract

Scheme 1

(a) Schematic illustration for the design of oxygen self-sufficient nanodroplet composed of fluorinated polymer (PDMAEMA-b-P(FMA-co-NMA)), perfluorocarbons (PFC) and AIE photosensitizer-TPA. (b) Therapeutic application of nanodroplet for animal dental caries model.

Fig. 1

Characterization of PFC/TPA@FNDs. (a) Hydrodynamic diameters distributions of PFC/TPA@FNDs; (b) TEM images of PFC/TPA@FNDs (Scale bar = 100 nm); (c) Absorbance spectra recorded for TPA, FNPs and PFC/TPA@FNDs; (d) Fluorescence emission spectra recorded for TPA in DMSO and PFC/TPA@FNDs in water, Exc: 445 nm; (e) Zeta potentials of PFC/TPA@FNDs in different pH solutions. (f) PFC loading amount (blue) and encapsulation efficiency (orange) of PFC/TPA@MNDs (fluorinated-free polymer) and PFC/TPA@FNDs; (g) Time dependent dissolved-oxygen concentrations of different samples. (h) ROS generation for O₂@PFC/TPA@FNDs, PFC/TPA@FNDs and TPA upon light irradiation (445 ± 5 nm, 56 mW/cm²) using ABDA as indicator, normalized absorbance intensity at 400 nm. (i) ¹O₂ generation of O₂@PFC/TPA@FNDs upon light irradiation (445 ± 5 nm, 56 mW/cm²) for different time evaluated by EPR spectrometer, TEMP as indicator.

Fig. 2

Bacteria adhesion and bacteria killing assays. (a) CLSM images of PFC/TPA@FNDs incubated with S. mutans for different time, green and pink stains indicate bacteria and PFC/TPA@FNDs, Scale bar = 5 μm; (b) TEM images of PFC/TPA@FNDs incubated with S. mutans for different time, respectively, Scale bar = 500 nm; (c) Bacterial viability calculated by bacterial colony formation after treatment with different concentrations of O₂@PFC/TPA@FNDs upon light irradiation or not (*** represent p < 0.001); (d) Typical agar plates of different concentration of O₂@PFC/TPA@FNDs incubated with S. mutans upon light irradiation (445 ± 5 nm, 56 mW/cm²) or not; (e) Fluorescence images of S. mutans upon treated with PBS or O₂@PFC/TPA@FNDs upon light irradiation (445 ± 5 nm, 56 mW/cm²) or not respectively, and then stained with SYTO 9 and PI, green and red stains indicate live bacteria and dead bacteria, Scale bar = 20 μm; (f) TEM images of S. mutans upon treated with PBS or O₂@PFC/TPA@FNDs and upon light irradiation or not respectively, Scale bar = 500 nm.

Fig. 3

Antibiofilm and biofilm penetration. (a) Typical agar plates of different samples incubate with S. mutans biofilm and then irradiate light or not using a plate counting method; (b) Bacterial colony counts of S. mutans biofilm after treatment with different samples upon irradiation light (445 ± 5 nm, 56 mW/cm²) or not (*** represent p < 0.001). (c) 2D and 3D CLSM images of different samples incubated with S. mutans biofilm upon irradiation light (445 ± 5 nm, 56 mW/cm²) or not, and then stained by SYTO 9 (green channel) and PI (red channel). The first and second columns refer to 2D bottom and middle CLSM images, and the third and fourth columns refer to 3D merge and 3D PI CLSM images, Scale bar = 15 μm; (d) Different magnification SEM images of S. mutans biofilm (in teeth) incubated with PBS, O₂@PFC/TPA@FNDs upon light irradiation or not, the red arrows refer to the disrupted bacterial morphology, Scale bar = 2 μm and 500 nm for top and down lines; (e) 2D and 3D CLSM images of PFC/TPA@FNDs incubate with S. mutans biofilm for different time, green and pink stains indicate bacteria and nanodroplets, Scale bar = 10 μm.

Fig. 4

Biosafety of nanodroplets. (a) Evaluation the cytotoxicity of different concentrations PFC/TPA@FNDs to Human Oral Keratinocytes cells (HOK); (b) Hemolysis ratio of different concentrations of PFC/TPA@FNDs, Inset: representative images for the hemolytic analysis.

Fig. 5

Animal dental caries model evaluating the therapeutic effect of nanodroplets. (a) Schematic illustration of the construction of dental caries animal model and the therapeutic process. (b) Images of basic fuchsin stained plaque after different treatments, yellow arrows showed the biofilm areas on the surface of tooth, Scale bar = 1 mm. (c) Different magnification SEM images of teeth in different groups after treatment, the microstructures of teeth surface were different, Scale bar = 100 μm and 2 μm respectively. (d) The cross-sectional view of Micro-CT results for teeth in different groups. Enamel thickness and caries depth were labeled by red line and yellow line respectively, and the numbers were calculated by CTAn software, Scale bar = 1 mm. (e) The summary of enamel thickness and caries depth in different groups, recorded by CTAn software (*, **, *** represent p < 0.05, p < 0.01 and p < 0.001).

Fig. 6

Tooth whitening effect of nanodroplets and biocompatibility. (a) Photographs of teeth-stained color and then treated with PBS, PFC/TPA@FNDs, 7.5 % H₂O₂, O₂@PFC/TPA@FNDs + Light for 0, 3, 5 h and cold-light whitening (30 min), respectively; (b)VITA classical shade guide for tooth color; (c)SEM images of healthy teeth and teeth treated with 7.5 % H₂O₂, 30 % H₂O₂, O₂@PFC/TPA@FNDs + Light, and cold-light whitening, Scale bar = 2 μm.