Toxicity evaluation of biodegradable chitosan nanoparticles using a zebrafish embryo model

Yu-Lan Hu¹, Wang Qi¹, Feng Han², Jian-Zhong Shao³, Jian-Qing Gao^1
1Institute of Pharmaceutics, College of Pharmaceutical Sciences, ²Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, ³College of Life Sciences, Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Zhejiang University, Hangzhou, Zhejiang, People's Republic of China

Background: Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In the present study, the zebrafish model was used for a safety evaluation of this nanocarrier.
Methods: Chitosan nanoparticles with two particle sizes were prepared by ionic cross-linking of chitosan with sodium tripolyphosphate. Chitosan nanoparticles of different concentrations were incubated with zebrafish embryos, and ZnO nanoparticles were used as the positive control.
Results: Embryo exposure to chitosan nanoparticles and ZnO nanoparticles resulted in a decreased hatching rate and increased mortality, which was concentration-dependent. Chitosan nanoparticles at a size of 200 nm caused malformations, including a bent spine, pericardial edema, and an opaque yolk in zebrafish embryos. Furthermore, embryos exposed to chitosan nanoparticles showed an increased rate of cell death, high expression of reactive oxygen species, as well as overexpression of heat shock protein 70, indicating that chitosan nanoparticles can cause physiological stress in zebrafish. The results also suggest that the toxicity of biodegradable nanocarriers such as chitosan nanoparticles must be addressed, especially considering the in vivo distribution of these nanoscaled particles.
Conclusion: Our results add new insights into the potential toxicity of nanoparticles produced by biodegradable materials, and may help us to understand better the nanotoxicity of drug delivery carriers.

Keywords: chitosan, nanoparticles, zebrafish embryo, nanotoxicology