Intracellular trafficking of silver nanoparticles and silver ions determined their specific mitotoxicity to the zebrafish cell line

Abstract

Silver nanoparticles (AgNPs) can penetrate cells and distribute in different organelles, resulting in adverse health effects. In the present study, for the first time, we quantitatively monitored AgNPs and Ag⁺ and identified the mitochondrial specific toxic mechanisms caused by the Ag⁺ released from AgNPs with different sizes and coating agents. Citrate coated AgNPs (Cit-AgNPs) with two different sizes and biocompatible aggregation-induced emission fluorogen (AIEgen) coated AgNPs (AIE-AgNPs) were employed in the present study. Bioimaging showed that more than 80% of Ag⁺, both the directly accumulated and dissolved Ag⁺ (derived from either Cit-AgNPs or AIE-AgNPs), was concentrated in the mitochondria as a major storage site and potential acting target in the ZF4 cells. By correlating between ICP-MS detection and flow cytometry analysis, the intracellular dissolution of AgNPs was 18.8%, 14.2% and 11.1% for Cit-AgNPs-1, Cit-AgNPs-2, and AIE-AgNPs, respectively, suggesting that the size and coating agents could affect the intracellular dissolution of AgNPs. Seahorse XFp analyzer measurements further showed that both AgNPs and Ag⁺ triggered mitochondrial specific toxicity, including deformation, lowered interconnectivity, and impaired respiration of mitochondria. The spare respiration capacity of cells decreased linearly with increasing dissolved Ag⁺ concentration derived from AgNPs with different sizes and coatings (coefficient, R² = 0.902), indicating the specific mitochondrial toxic effects caused by the dissolved Ag⁺ derived from AgNPs with different sizes and coating agents. Our results strongly suggested that AgNP toxicity was attributed to dissolved Ag⁺, which was released, transported, and concentrated in the mitochondria, finally leading to exhaustion of the reserve respiratory capacity and cell death.