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A general mechanism for intracellular toxicity of metal-containing nanoparticles.

Sabella S, Carney RP, Brunetti V, Malvindi MA, Al-Juffali N, Vecchio G, Janes SM, Bakr OM, Cingolani R, Stellacci F, Pompa PP - Nanoscale (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Istituto Italiano di Tecnologia, Center for Bio-Molecular Nanotechnologies@UniLe, Via Barsanti, 73010 Arnesano (Lecce), Italy. pierpaolo.pompa@iit.it.

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.

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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.
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fig5: 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.

Mentions: We verified our model on several types of metal containing NPs (see Table S32† for a complete list of tested NPs and their relative physical–chemical characterization), observing that the toxicity can be mainly ascribed to that of the corresponding ions. First, as shown in Fig. 5, we probed the toxicity of the four types of NPs previously tested (i.e., Au, Ag, Fe3O4, and CdSe/ZnS NPs) upon incubation with specific ion chelators.45,46 It is noteworthy that, for all the NPs tested, the presence of the chelating agents leads to a significant reduction of the NP toxicity. This indicates a major role of intracellularly released ions in eliciting NP toxicity. When the toxic ions are chelated, the toxic effects of the NPs are minimized. Note that, in the conditions employed in these experiments, the use of chelators did not affect the uptake efficiency of the tested NPs (see Fig. S18†), nor did it induce spurious off-target effects on cells (as shown in a “cross-toxicity test”, Fig. S19,† using specific and aspecific chelating molecules). Interestingly, and in agreement with previous studies,22 the ROS level and the cellular membrane integrity have also been found to be altered by NP treatments (Fig. S20†). Yet, these toxic outcomes are also drastically reduced by the presence of chelants, further demonstrating the central role of the in situ released ions in mediating cellular toxicity (see also below).


A general mechanism for intracellular toxicity of metal-containing nanoparticles.

Sabella S, Carney RP, Brunetti V, Malvindi MA, Al-Juffali N, Vecchio G, Janes SM, Bakr OM, Cingolani R, Stellacci F, Pompa PP - Nanoscale (2014)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120234&req=5

fig5: 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.
Mentions: We verified our model on several types of metal containing NPs (see Table S32† for a complete list of tested NPs and their relative physical–chemical characterization), observing that the toxicity can be mainly ascribed to that of the corresponding ions. First, as shown in Fig. 5, we probed the toxicity of the four types of NPs previously tested (i.e., Au, Ag, Fe3O4, and CdSe/ZnS NPs) upon incubation with specific ion chelators.45,46 It is noteworthy that, for all the NPs tested, the presence of the chelating agents leads to a significant reduction of the NP toxicity. This indicates a major role of intracellularly released ions in eliciting NP toxicity. When the toxic ions are chelated, the toxic effects of the NPs are minimized. Note that, in the conditions employed in these experiments, the use of chelators did not affect the uptake efficiency of the tested NPs (see Fig. S18†), nor did it induce spurious off-target effects on cells (as shown in a “cross-toxicity test”, Fig. S19,† using specific and aspecific chelating molecules). Interestingly, and in agreement with previous studies,22 the ROS level and the cellular membrane integrity have also been found to be altered by NP treatments (Fig. S20†). Yet, these toxic outcomes are also drastically reduced by the presence of chelants, further demonstrating the central role of the in situ released ions in mediating cellular toxicity (see also below).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Istituto Italiano di Tecnologia, Center for Bio-Molecular Nanotechnologies@UniLe, Via Barsanti, 73010 Arnesano (Lecce), Italy. pierpaolo.pompa@iit.it.

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.

Show MeSH
Related in: MedlinePlus