Intracellular trafficking pathways in silver nanoparticle uptake and toxicity in Caenorhabditis elegans

Abstract

We used the nematode Caenorhabditis elegans to study the roles of endocytosis and lysosomal function in uptake and subsequent toxicity of silver nanoparticles (AgNP) in vivo. To focus on AgNP uptake and effects rather than silver ion (AgNO3) effects, we used a minimally dissolvable AgNP, citrate-coated AgNPs (CIT-AgNPs). We found that the clathrin-mediated endocytosis inhibitor chlorpromazine reduced the toxicity of CIT-AgNPs but not AgNO3. We also tested the sensitivity of three endocytosis-deficient mutants (rme-1, rme-6 and rme-8) and two lysosomal function deficient mutants (cup-5 and glo-1) as compared to wild-type (N2 strain). One of the endocytosis-deficient mutants (rme-6) took up less silver and was resistant to the acute toxicity of CIT-AgNPs compared to N2s. None of those mutants showed altered sensitivity to AgNO3. Lysosome and lysosome-related organelle mutants were more sensitive to the growth-inhibiting effects of both CIT-AgNPs and AgNO3. Our study provides mechanistic evidence suggesting that early endosome formation is necessary for AgNP-induced toxicity in vivo, as rme-6 mutants were less sensitive to the toxic effects of AgNPs than C. elegans with mutations involved in later steps in the endocytic process.

Keywords: In vivo endocytosis; lysosome; nanoparticle uptake; nanotoxicology.

Figure 1

Dose response curves for ionic silver and AgNP toxicity with and without chlorpromazine (10 mg L⁻¹). A) AgNO₃ (0.025–0.1 mg-Ag L⁻¹); B) CIT-AgNPs (0.1–1.5 mg-Ag L⁻¹).

Figure 2

A) Schematic illustration of endocytosis-related uptake pathways for NPs and all the mutants utilized in this study. (B–C) Percent mortality of N2 and endocytosis mutants 24 h post exposure to (B) AgNO₃ (0.05 mg-Ag L⁻¹) and (C) citrate-coated AgNP (CIT-AgNPs) (1 mg-Ag L⁻¹). *indicates that the strain was significantly different from N2 (Kruskal-Wallis). For AgNO₃ exposure, N= 18 for each strain, with data pooled from 3 separate experiments. For CIT-AgNP exposure, N= 12 for each strain, with data pooled from 2 separate experiments. Boxplots show the 10%, 25%, median, 75% and 90% quantiles for mortality. D) Total silver content (+/− standard error of the mean) in all strains upon exposure to AgNO₃ and CIT-AgNPs, as measured by ICP-MS. For each strain, N=5 for AgNO₃ and N=6 for CIT-AgNPs, and data were pooled from 2 separate batches of nematode samples. E) Correlation between mean mortality 24 h after the silver treatments and total silver concentration in nematodes based on nematode dry weight.

Figure 3

Percent mortality of N2 and lysosomal mutants 24 hours post exposure to (A) AgNO₃ (0.0 – 0.12 mg-Ag L⁻¹) and (B) CIT-AgNPs (0.0 – 1.0 mg-Ag L⁻¹). Data represent 4–6 replicate experiments, with each experiment containing 6 replicate wells. No statistically significant strain differences in response to AgNO3 or CIT-AgNPs were detected (p>0.05 for significance of interaction term in 2-factor ANOVA).