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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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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 ESI†1). 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),31,32 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.
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fig1: 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 ESI†1). 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),31,32 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.

Mentions: First, we investigated the time-dependent ion release of a variety of NPs, namely metallic (Au and Ag), magnetic (Fe3O4) and semiconductor (CdSe/ZnS) NPs. The synthesis and the physico-chemical characterizations of these NPs are reported in the Methods section in the ESI.† The ion leakage from the NPs was assessed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) under two separate conditions, mimicking either the lysosomal environment (37 °C, pH 4.5)31,32 or the cellular cytoplasmic environment (37 °C, neutral pH) (see Methods section in the ESI† for details). As shown in Fig. 1, for all NPs tested, we observed significant ion release in the acidic conditions and no measurable release in neutral conditions (the NP behavior in neutral conditions was also tested in cell culture medium, DMEM, 10% FBS, pH 7.4, as a more relevant model of physiological conditions, and the same results were obtained). Such ion release was accompanied by obvious NP degradation, with consequent loss of NP morphology as well as of their fluorescence or magnetic properties (in the case of CdSe/ZnS or Fe3O4, respectively, see Fig. 1, bottom) (a more detailed analysis of NP degradation in an acidic environment is reported in the ESI, Fig. S1–S4†). The ion release profiles were specific to the NPs under investigation, depending on their core material, initial concentration, and specific coating. In any case, we found that the lysosomal environment is capable of promoting NP degradation/corrosion. We hypothesized that such ions, once released intracellularly, are likely to be the main factor in promoting the toxic effects of NPs (see 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)

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 ESI†1). 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),31,32 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: 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 ESI†1). 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),31,32 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.
Mentions: First, we investigated the time-dependent ion release of a variety of NPs, namely metallic (Au and Ag), magnetic (Fe3O4) and semiconductor (CdSe/ZnS) NPs. The synthesis and the physico-chemical characterizations of these NPs are reported in the Methods section in the ESI.† The ion leakage from the NPs was assessed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) under two separate conditions, mimicking either the lysosomal environment (37 °C, pH 4.5)31,32 or the cellular cytoplasmic environment (37 °C, neutral pH) (see Methods section in the ESI† for details). As shown in Fig. 1, for all NPs tested, we observed significant ion release in the acidic conditions and no measurable release in neutral conditions (the NP behavior in neutral conditions was also tested in cell culture medium, DMEM, 10% FBS, pH 7.4, as a more relevant model of physiological conditions, and the same results were obtained). Such ion release was accompanied by obvious NP degradation, with consequent loss of NP morphology as well as of their fluorescence or magnetic properties (in the case of CdSe/ZnS or Fe3O4, respectively, see Fig. 1, bottom) (a more detailed analysis of NP degradation in an acidic environment is reported in the ESI, Fig. S1–S4†). The ion release profiles were specific to the NPs under investigation, depending on their core material, initial concentration, and specific coating. In any case, we found that the lysosomal environment is capable of promoting NP degradation/corrosion. We hypothesized that such ions, once released intracellularly, are likely to be the main factor in promoting the toxic effects of NPs (see 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