A general mechanism for intracellular toxicity of metal-containing nanoparticles

Abstract

The assessment of the risks exerted by nanoparticles is a key challenge for academic, industrial, and regulatory communities worldwide. Experimental evidence points towards significant toxicity for a range of nanoparticles both in vitro and in vivo. Worldwide efforts aim at uncovering the underlying mechanisms for this toxicity. Here, we show that the intracellular ion release elicited by the acidic conditions of the lysosomal cellular compartment--where particles are abundantly internalized--is responsible for the cascading events associated with nanoparticles-induced intracellular toxicity. We call this mechanism a "lysosome-enhanced Trojan horse effect" since, in the case of nanoparticles, the protective cellular machinery designed to degrade foreign objects is actually responsible for their toxicity. To test our hypothesis, we compare the toxicity of similar gold particles whose main difference is in the internalization pathways. We show that particles known to pass directly through cell membranes become more toxic when modified so as to be mostly internalized by endocytosis. Furthermore, using experiments with chelating and lysosomotropic agents, we found that the toxicity mechanism for different metal containing NPs (such as metallic, metal oxide, and semiconductor NPs) is mainly associated with the release of the corresponding toxic ions. Finally, we show that particles unable to release toxic ions (such as stably coated NPs, or diamond and silica NPs) are not harmful to intracellular environments.

Fig. 1. Time-dependent ion release, probed by ICP-AES, of different NPs at 37 °C, in neutral (blue symbols) or acidic (red symbols) conditions. (A) 4 nm AuNPs, 50 nM concentration (striped and unstructured AuNPs showed similar behavior, see also Fig. S5†); (B) 5 nm AgNPs, 17 nM concentration; (C) 6 nm CdSe/ZnS NPs, 20 nM concentration; (D) 10 nm Fe₃O₄ NPs, 40 nM concentration. The reported sizes of the NPs refer to their core structures (see Table S32 in the ESI1). In (C) and (D) bottom, representative photographs of the respective NPs are also shown, at time 0 and after 96 h in acidic conditions, clearly revealing a significant loss of NPs' fluorescence (C) or magnetic (D) properties after the acidic treatment. Neutral and acidic conditions were obtained by dispersing the NPs in water (pH 7.0) or in citrate buffer (pH 4.5),^, respectively. Neutral conditions were also probed in cell culture medium (DMEM, 10% FBS, pH 7.4), obtaining the same results (i.e., no detectable ion release). Data represent the average from 3 independent measurements (6 replicates for each experiment) and the error bars indicate the standard deviation.

Fig. 2. Toxicity assessment of striped and unstructured AuNPs in U937 cells. (A) WST-8 proliferation assay upon treatment with increasing amount of AuNPs. Ctrl represents the negative control; values are mean ± SD. Positive controls (not shown) were treated with 0.01% of TritonX100, displaying a strong viability decrease (ca. 80–90%) with respect to the untreated cells. (B) ROS quantification, via DCFH-DA assay, after cellular treatment with AuNPs; values are mean ± SD. Positive controls (not shown) were treated with a free radical generator (100 μM H₂O₂), exhibiting a ROS increase of ca. 190–220% with respect to the untreated control cells. (C) Evaluation of caspase 3 activity. Values are mean ± SD. Results were analyzed by Two-way ANOVA and values compared to the control by the Bonferroni post-hoc test. Differences between treated samples and controls (n = 8) were considered statistically significant for ***P < 0.001, **P < 0.01, *P < 0.05, and non-significant for P > 0.05.

Fig. 3. Inhibition of thioredoxin reductase (TrxR) by AuNPs. (A) Inhibition of TrxR activity by the gold ions released by AuNPs at different pH values (pH 4.5 or 7.0), according to the experiment reported in Fig. 1. The inhibitor drug auranofin was used as a positive control (see Methods section in the ESI†), while gold ion concentration at pH 4.5 was 20 nM (gold ion release was not detectable at pH 7.0). (B) Cellular TrxR activity in HeLa cells after 48 h treatment with striped and unstructured AuNPs (15 nM) and auranofin (1 μM) (see Methods section in the ESI for experimental details). Results are mean ± SD and differences between treated samples and controls (n = 8) were considered statistically significant for ***P < 0.001.

Fig. 4. ICP-AES of intracellular gold ions released by striped and unstructured AuNPs upon internalization by HeLa cells. Cells were treated with striped and unstructured AuNPs (20 nM) for 48 hours. Upon incubation, cells were lysed and ultrafiltrated by Amicon Ultra-4 (experimental details are reported in the Methods section in the ESI†). The filtered solutions containing all the soluble ions were then analyzed by ICP-AES. The amount of gold ions is reported as μmol of filtered gold ions per cell. Results show that striped AuNPs do not release a measurable amount of ions, whereas unstructured AuNPs, being entrapped in the lysosomes, are subjected to partial acidic corrosion, leading to the release of their ion cargo.

Fig. 5. Toxicity assessment of different types of NPs in the absence/presence of specific ion chelators. WST-8 proliferation assays upon treatment with (A) striped and unstructured AuNPs (20 nM), (B) AgNPs (2 nM), and (C) CdSe/ZnS QDs (5 nM) in HeLa cells in the presence of 2,3-dithiopropanol (BAL); HeLa cells were pretreated for 30 min with/without 1 μM BAL and then exposed to the NPs for 24–48 h. (D) Proliferation assay upon treatment with 2.5 nM of Fe₃O₄ NPs. In this case, HeLa cells were pretreated for 30 min with/without 100 μM desferrioxamine (dfx) and then exposed to Fe₃O₄ NPs for 24–48 h. In all cases, the pretreatment with chelating agents suppresses, almost totally, the toxicity of the NPs. CTRL represents the negative control; values are mean ± SD. Differences between treated samples and controls (n = 8) were considered statistically significant for *P < 0.05 and non-significant for P > 0.05.

Fig. 6. Toxicity assessment of unstructured AuNPs and Fe₃O₄ NPs in HeLa cells, in the presence/absence of two different lysosomotropic agents (chloroquine or ammonium chloride). (A and B) WST-8 proliferation assay upon treatment with Fe₃O₄ NPs (2.5 nM) and (C and D) unstructured AuNPs (20 nM). HeLa cells were pretreated for 30 min with/without 5 μM chloroquine (panels A and C) or 5 mM ammonium chloride (panels B and D) and then exposed to the NPs for 48 h. In all cases, NPs toxicity was strongly attenuated by the lysosomotropic agents that neutralize lysosomal acidity, preventing ion release and cytotoxicity. CTRL represents the negative control; values are mean ± SD. Differences between treated samples and controls (n = 8) were considered statistically significant for *P < 0.05 and non-significant for P > 0.05.

Fig. 7. Schematic of the general toxicity mechanism induced by NPs when they enter cells by active internalization mechanisms, compared to endocytosis-free NPs. NPs that enter the cells by energy-dependent processes (mediated by clathrin, caveolin, lipid raft formations and others) are rapidly confined in vesicular structures, endosomes, and finally in lysosomes. The acidic lysosomal pH triggers a lysosome-enhanced Trojan horse effect (LETH effect) that combines the abundant cellular internalization of the NPs via active processes with the consequent enhanced release of the relatively toxic ions (e.g., Ag⁺, Cd²⁺, Fe^2+/3+, Au^1+/3+ ions). The significant amount of intracellularly leaked ions may then exert ion-specific toxicity (e.g., enzyme depletion/inactivation, protein denaturation, etc.) against some cellular targets (e.g., mitochondria, RER) and/or lysosomal damage/dysfunction. This finally results in increased ROS levels, apoptosis, DNA and membrane damage.