Skin Toxicity Assessment of Silver Nanoparticles in a 3D Epidermal Model Compared to 2D Keratinocytes

• Main Authors: Chen L, Wu M, Jiang S, Zhang Y, Li R, Lu Y, Liu L, Wu G, Liu Y, Xie L, Xu L
• Format: Article
• Language: English
• Published: Dove Medical Press 2019-12-01
• Series: International Journal of Nanomedicine
• Subjects: silver nanoparticles (agnps); cytotoxicity; keratinocytes; 3d epidermal model; epikutis®; oxidative stress; inflammatory response
• Online Access: https://www.dovepress.com/skin-toxicity-assessment-of-silver-nanoparticles-in-a-3d-epidermal-mod-peer-reviewed-article-IJN

Liang Chen,1,* Meiyu Wu,2,* Shan Jiang,1,3,* Yanyun Zhang,4 Runzhi Li,4 Yongbo Lu,4 Lin Liu,2 Gang Wu,3 Ying Liu,5 Liming Xie,2 Liming Xu1 1NCNST-NIFDC Joint Laboratory for Measurement and Evaluation of Nanomaterials in Medical Applications, National Institutes for Food and Drug Control, Beijing 102629, People’s Republic of China; 2CAS Key Laboratory of Standardization and Measurement for Nanotechnology, NCNST-NIFDC Joint Laboratory for Measurement and Evaluation of Nanomaterials in Medical Applications, Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, People’s Republic of China; 3Department of Preclinical Medicine and Forensic, Baotou Medical College, Baotou 014040, People’s Republic of China; 4Guangdong Biocell Biotechnology Co. Ltd, Dongguan, 523808, Guangdong, People’s Republic of China; 5CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, NCNST-NIFDC Joint Laboratory for Measurement and Evaluation of Nanomaterials in Medical Applications, Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, People’s Republic of China*These authors contributed equally to this workCorrespondence: Liming XuNCNST-NIFDC Joint Laboratory for Measurement and Evaluation of Nanomaterials in Medical Applications, National Institutes for Food and Drug Control, Beijing, People’s Republic of ChinaEmail [e-mail] XieCAS Key Laboratory of Standardization and Measurement for Nanotechnology, NCNST-NIFDC Joint Laboratory for Measurement and Evaluation of Nanomaterials in Medical Applications, Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, People’s Republic of ChinaEmail [e-mail]: Increased use of silver nanoparticles (AgNPs) has raised concerns that AgNPs may induce toxic effects. In vitro studies of cell monolayers and in vivo studies have produced conflicting results. The inconsistency of these results has been mainly due to limitations of two-dimensional (2D) monolayer cell systems.Methods: A three-dimensional (3D) epidermal model called EpiKutis®, which exhibits good tissue viability and barrier function was developed. The cytotoxicity of AgNPs against EpiKutis was compared to that against 2D keratinocytes at equivalent AgNPs doses (0.035, 0.07, 0.14, 0.28, and 0.56 ng per cell). The amount and distribution of AgNPs in the 3D EpiKutis and 2D keratinocytes after exposure were determined. The toxic mechanisms of AgNPs, such as oxidative stress and production of pro-inflammatory cytokines, were investigated.Results: The results demonstrated that cell viability was greater than 80% and lactate dehydrogenase (LDH) release did not increase even at the highest dose of AgNPs in EpiKutis. In contrast, treatment of 2D keratinocytes with AgNPs resulted in dose-dependent decrease in cell viability from 63% to 11%, and a dose-dependent increase in LDH release from 8% to 16%. Cytotoxicity of AgNPs in 2D keratinocytes was related to oxidative damage and inflammation, as evidenced by increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), IL-1α, IL-6, and IL-8. In addition, levels of superoxide dismutase (SOD) were decreased. EpiKutis treated with AgNPs did not exhibit increased oxidative damage or inflammation, which may have been due to the barrier properties of the 3D structure, resulting in reduced penetration of AgNPs. At equivalent per cell doses, total silver penetration into EpiKutis was 0.9 ± 0.1%, and total silver penetration into 2D keratinocytes was 8.8 ± 0.6% detected by ICP-MS. The penetration and distribution of AgNPs in 2D keratinocytes were confirmed by the TEM-EDS analysis, which was not found in the 3D EpiKutis. These results showed that AgNPs penetrated EpiKutis to a lesser degree than they penetrated 2D keratinocytes, which suggested that EpiKutis exhibited significant barrier function.Discussion: The results of this study showed that AgNP toxicity should be evaluated using 3D epidermal models, which may provide better estimates of in vivo conditions than 2D models. The EpiKutis model may be an ideal model for assessment of nanotoxicity.Keywords: silver nanoparticles, AgNPs, cytotoxicity, keratinocytes, 3D epidermal model, EpiKutis®, oxidative stress, inflammatory response