Development of a Chitosan-Based Nanoemulsion for Mitigating Microplastic-Induced Cytotoxicity and Oxidative Stress in RTgillW1 Fish Gill Cells

Abstract

Microplastic pollution is an emerging environmental concern that induces oxidative stress, inflammation, and cellular injury in aquatic organisms. Despite increasing evidence of microplastic toxicity, effective mitigation strategies remain limited. This study investigated the protective effects of a chitosan-based nanoemulsion (CS-NE) against polystyrene microplastic (PS-MP)-induced cytotoxicity and oxidative stress in RTgill-W1 fish gill cells. Chitosan nanoemulsions were prepared using ionic gelation-emulsification assisted by ultrasonication and characterized by particle size, polydispersity index (PDI), and zeta potential analyses. RTgill-W1 cells were exposed to PS-MPs (100 µg/mL) alone or in combination with low-dose (50 µg/mL) or high-dose (100 µg/mL) CS-NE for 24 h. Cell viability, oxidative stress biomarkers, and inflammatory gene expression were evaluated. The developed nanoemulsion exhibited a mean particle size of 118.6 ± 4.3 nm, a PDI of 0.21 ± 0.02, and a zeta potential of +34.8 ± 2.1 mV, indicating good colloidal stability. High-dose CS-NE showed significantly greater microplastic-binding efficiency (78.5 ± 3.2%) than the low-dose formulation (52.8 ± 2.7%). Exposure to PS-MPs reduced cell viability from 100.0 ± 2.8% to 49.2 ± 3.6%, whereas high-dose CS-NE restored viability to 91.6 ± 3.4%. PS-MPs significantly increased reactive oxygen species (276 ± 14%) and malondialdehyde levels (7.4 ± 0.6 nmol/mg protein), while reducing catalase activity and glutathione content. Co-treatment with high-dose CS-NE markedly reduced ROS (116 ± 8%) and MDA (2.8 ± 0.3 nmol/mg protein) while restoring catalase (46.2 ± 2.3 U/mg protein) and GSH (7.5 ± 0.4 µmol/g protein) levels. Furthermore, CS-NE suppressed TNF-α (1.32 ± 0.12-fold) and IL-1β (1.48 ± 0.13-fold) expression and enhanced Nrf2 expression (1.21 ± 0.09-fold). These findings demonstrate that chitosan-based nanoemulsions mitigate microplastic-induced cellular injury through microplastic sequestration and modulation of oxidative stress and inflammatory pathways, highlighting their potential as a novel nanotechnological intervention for aquatic environmental protection.