Photodynamic and nitric oxide therapy-based synergistic antimicrobial nanoplatform: an advanced root canal irrigation system for endodontic bacterial infections

Abstract

Background: The main issues faced during the treatment of apical periodontitis are the management of bacterial infection and the facilitation of the repair of alveolar bone defects to shorten disease duration. Conventional root canal irrigants are limited in their efficacy and are associated with several side effects. This study introduces a synergistic therapy based on nitric oxide (NO) and antimicrobial photodynamic therapy (aPDT) for the treatment of apical periodontitis.

Results: This research developed a multifunctional nanoparticle, CGP, utilizing guanidinylated poly (ethylene glycol)-poly (ε-Caprolactone) polymer as a carrier, internally loaded with the photosensitizer chlorin e6. During root canal irrigation, the guanidino groups on the surface of CGP enabled effective biofilm penetration. These groups undergo oxidation by hydrogen peroxide in the aPDT process, triggering the release of NO without hindering the production of singlet oxygen. The generated NO significantly enhanced the antimicrobial capability and biofilm eradication efficacy of aPDT. Furthermore, CGP not only outperforms conventional aPDT in eradicating biofilms but also effectively promotes the repair of alveolar bone defects post-eradication. Importantly, our findings reveal that CGP exhibits significantly higher biosafety compared to sodium hypochlorite, alongside superior therapeutic efficacy in a rat model of apical periodontitis.

Conclusions: This study demonstrates that CGP, an effective root irrigation system based on aPDT and NO, has a promising application in root canal therapy.

Keywords: Antimicrobial photodynamic therapy; Antimicrobials; Biofilm; Multifunctional nanoparticles; Nitric oxide gas therapy; Osteogenesis; Root canal irrigation.

Scheme 1

CGP preparation schematic and synergistic PDT/NO antibacterial effect. Schematic representation of CGP preparation, accompanied by an elucidation of the mechanisms underlying the highly efficient synergistic antibacterial action of PDT/NO and its role in promoting apical periodontitis healing

Fig. 1

Fabrication and characterization of CGP nanoparticles. a Schematic illustration for the preparation of CGP nanoparticles. b The particle size distribution and c transmission electron microscope images of CPP. d The particle size distribution and e transmission electron microscope images of CGP. f Solubility of Ce6 in different groups. g Zeta potential of each group. h Stability of CGP during 14 d. i Free Ce6, CPP, and CGP internalized by or bound tightly to E. faecalis and j the quantitative fluorescence intensity of Ce6 in E. faecalis. k Confocal laser scanning microscope images of E. faecalis biofilms treated by various groups. Data are presented as mean ± SEM, n = 3, *p < 0.05, **p ≤ 0.01, ****p ≤ 0.0001. Comparison between CGP versus other groups

Fig. 2

NO and ROS generation profiles of CGP. a NO, b H₂O₂, and c ¹O₂ generation profiles of each group. d, e NO, f, g ROS, and h, i H₂O₂ generation and mean fluorescence intensity in E. faecalis bacteria after receiving various treatments. Data are presented as mean ± SEM, n = 3, **p ≤ 0.01, ****p ≤ 0.0001, ns: no significance. Comparison between CGP + Laser versus other groups

Fig. 3

In vitro antibacterial activity of CGP. a Representative images of plate samples and b bacterial viability, c Live/Dead staining, and d scanning electron microscope images of E. faecalis after receiving various treatments. e The amount of total protein released from E. faecalis and f intracellular adenosine 5'-triphosphate production levels in E. faecalis after receiving various treatments. The red arrows point to the holes in cell membranes. The red arrows point to the damaged bacterial membranes. Data are presented as mean ± SEM. In (b) n = 3, while in (e, f) n = 5. **p ≤ 0.01, ****p ≤ 0.0001, ns: no significance. In (b) comparison between PBS versus other groups, while in (e, f) between the CGP + Laser versus other groups

Fig. 4

In vitro biofilm eradication efficacy of CGP. a Live/Dead staining, b crystalline violet staining, and c residual rates of E. faecalis biofilms after receiving various treatments. d Sample plate representative images, e bacterial viability, and f scanning electron microscope images of E. faecalis biofilms in human extracted teeth after receiving various irrigations. Data are presented as mean ± SEM, n = 3, *p ≤ 0.05, ****p ≤ 0.0001, ns: no significance. Comparison between 1% NaClO versus other groups

Fig. 5

Blood compatibility and biocompatibility of CGP. a Microscopic images of erythrocyte aggregation in different treatment groups. b, c Hemolytic effects in different treatment groups. d Cell Counting Kit-8 assay of MC3T3-E1 and e hPDLSCs cells after treated with various groups. f Live/dead cell staining in different groups of hPDLSCs. Data are presented as mean ± SEM. In (c) n = 3, while in (d, e) n = 5. *p < 0.05, ***p ≤ 0.001, ****p ≤ 0.0001, ns: no significance. In (c) comparison between PBS versus other groups, while in (d, e) between the control group versus other groups

Fig. 6

Mechanisms of CGP promoting repair of periapical bone defects. a, b Images and quantitative data of ALP staining and c, d alizarin red staining in MC3T3-E1 cells after receiving various treatments in mineralizing conditions. e The expression levels of OCN and f RUNX2 in MC3T3-E1 cells after receiving various treatments in mineralizing conditions. Data are presented as mean ± SEM, n = 3, *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns: no significance. Comparison between H₂O₂ + CGP versus other groups

Fig. 7

In vivo therapeutic efficacy of CGP apical periodontitis model rats. a Experimental design of apical periodontitis model rats. b, c Sample plate representative images and bacterial viability of E. faecalis in rat teeth after receiving various irrigations on day 0 and d, e day 21. f Representative images and g the total resorption volume of the left maxillary first molar in each group. h Representative HE staining and i TRAP staining. The green arrows point to the neutrophils and the red arrows point to active osteoclasts. Data are presented as mean ± SEM, n = 6, ****p ≤ 0.0001. Comparison between PBS versus other groups