Genotoxicity Induced by Cellular Uptake of Chitosan Nanoparticles in Human Dental Pulp Cells

Objective: Recent in vitro studies have shown that chitosan nanoparticles in several root canal sealers, intracanal medicament, and irrigation solutions could enhance the antimicrobial activity. However, the nanotoxicity of chitosan has not been fully studied. The aim of this study was to evaluate cellular uptake and genotoxicity of various sizes and concentrations of chitosan nanoparticles cultured with human dental pulp cells. Methods: Human dental pulp cells were derived from human dental pulp tissues and cultured for 24 hours with 50 nm and 318 nm FITC-tagged chitosan nanoparticles in concentrations: 0.1 mg/mL, 0.5 mg/mL, and 2 mg/mL as study groups, and 0 mg/mL as a control. The fluorescence intensity of the FITC tagged chitosan nanoparticles was measured using a spectrophotometer to determine the cellular uptake. Genotoxicity was assessed by the Cytokinesis-block micronucleus method and by measuring the fluorescent intensity of the phosphorylated H2AX nuclear foci. Statistical analysis was performed using One-Way ANOVA, post-hoc Tukey, and Chi-square tests. Results: Chitosan nanoparticles were able to internalize the human dental pulp cells and significantly induced micronuclei, nuclear buds, and pH2AX foci at concentrations of 0.5 mg/mL and 2 mg/mL as compared to 0.1 mg/mL (P < 0.01) and control group (P < 0.01). At both concentrations, 0.5 mg/mL and 2 mg/mL, 50 nm chitosan significantly induced higher proportions of micronuclei (P=0.001), nuclear buds (P=0.009), and pH2AX nuclear foci (P=0.00004) as compared to 318 nm chitosan. Conclusion: 50 nm and 318 nm chitosan nanoparticles at concentrations 0.5 mg/mL and 2 mg/mL penetrated human dental pulp cells and induced genotoxicity in dose-dependent and size-associated manners.