A combined proteomics and metabolomics approach to assess the effects of gold nanoparticles in vitro

Omics technologies, such as proteomics or metabolomics, have to date been applied in the field of nanomaterial safety assessment to a limited extent. To address this dearth, we developed an integrated approach combining the two techniques to study the effects of two sizes, 5 and 30?nm, of gold nanoparticles (AuNPs) in Caco-2 cells. We observed differences in cells exposed for 72?h to each size of AuNPs: 61 responsive (up/down-regulated) proteins were identified and 35 metabolites in the cell extract were tentatively annotated. Several altered biological pathways were highlighted by integrating the obtained multi-omics data with bioinformatic tools. This provided a unique set of molecular information on the effects of nanomaterials at cellular level. This information was supported by complementary data obtained by immunochemistry, microscopic analysis, and multiplexed assays. A part from increasing our knowledge on how the cellular processes and pathways are affected by nanomaterials (NMs), these findings could be used to identify specific biomarkers of toxicity or to support the safe-by-design concept in the development of new nanomedicines.     

Size-dependent toxicity and cell interaction mechanisms of gold nanoparticles on mouse fibroblasts

Gold nanoparticles (AuNPs) are currently used in several fields including biomedical applications, although no conclusive information on their cytotoxicity is available. For this reason this work has investigated the effects of AuNPs in vitro on Balb/3T3 mouse fibroblasts. Results obtained exposing cells for 72 h to AuNPs 5 and 15 nm citrate stabilized, revealed cytotoxic effects only for AuNPs 5 nm at concentration ≥ 50 μM if measured by colony forming efficiency (CFE). To understand the differences in cytotoxicity observed for the two AuNPs sizes, we investigated the uptake and the intracellular distribution of the nanoparticles. By TEM it was observed that 5 and 15 nm AuNPs are internalized by Balb/3T3 cells and located within intracellular endosomal compartments. Quantification of the uptake by ICP-MS showed that AuNPs internalization enhanced even up to 72 h. Disruption of the actin cytoskeleton was evident, with cell footprints narrow and contracted; effects more remarkable in cells exposed to 5 nm AuNP. The mechanism of NPs cell internalization was investigated using immunocytochemistry and western blot. No significant effect was observed in the expression level of caveolin, while reduction of the expression and degradation of the clathrin heavy chain was observed in cells exposed for 72 h to AuNPs.     

A quantitative in vitro approach to study the intracellular fate of gold nanoparticles: from synthesis to cytotoxicity

Abstract

Due to their physico-chemical characteristics, gold nanoparticles (AuNPs) seem to be suitable for biomedical and therapeutic applications even if conflicting data on their toxicological profiles are present in literature. In order to better understand if AuNPs could be safe we must consider different biological endpoints such as cytotoxicity, genotoxicity, inflammation and biopersistence. Starting from these considerations, one of the first issues to be assessed is to better understand if AuNPs can be internalized by cells. In this work, we propose a methodological approach to radioactivate AuNPs by neutron activation and the quantification of their internalization by two in vitro cell systems such as MDCK and HepG2 after 24 h of exposure. Despite a dose-dependent internalization, no evidence of cytotoxicity, determined by two different standard in vitro methods such as Neutral Red Uptake and Colony Forming Efficiency, was observed.     

The adsorption of cellular proteins affects the uptake and cellular distribution of gold nanoparticles

Nanotechnology has been widely used in the field of medicine, and it can significantly improve the bioavailability and the target efficiency of medicines. However, after administration, nanomedicines can adsorb biomolecules that can influence their effects. It was reported that the adsorption of plasma proteins can change the surface properties of nanoparticles. When nanoparticles pass through cells, they may carry some cellular proteins out of cells. Currently, it is unclear whether the adsorbed proteins affect the uptake of nanoparticles in the next cell layer. To simplify this complex biological process, BSA-capped gold nanoparticles were prepared and incubated with Caco-2 cell lysate to simulate conditions of transcytosis through epithelial cells. The surface morphology of nanoparticles was examined by TEM. SRB was used to evaluate the cytotoxicity of the nanoparticles. The uptake and cellular distribution of the nanoparticles were detected by ICP-MS and CLSM. The results suggested that the adsorption of cell proteins could enhance the adhesion and uptake of gold nanoparticles. The gold nanoparticles were mainly located in lysosomes, and there were some Lysate-capped AuNPs in the mitochondria whereas no BSA-capped AuNPs appeared there.     

Cellular uptake and toxicity of gold nanoparticles on two distinct hepatic cell models

Gold nanoparticles (AuNPs) have huge potential for various biomedical applications, but their successful use depends on their uptake and possible toxicity in the liver, their main site for accumulation. Therefore, in this work we compared the cytotoxic effects induced by AuNPs with different size (~ 15 nm and 60 nm), shape (nanospheres and nanostars) and capping [citrate- or 11-mercaptoundecanoic acid (MUA)], in human HepaRG cells or primary rat hepatocytes (PRH) cultivated with serum-free or Foetal Bovine Serum (FBS)-supplemented media.The safety assessment of the AuNPs demonstrated that overall they present low toxicity towards hepatic cells. Among all the tested AuNPs, the smaller 15 nm spheres displayed the highest toxicity. The toxicological effect was capping, size and cell-type dependent with citrate-capping more toxic than MUA (PRH with FBS), the 15 nm AuNPs more toxic than 60 nm counterparts and PRH more sensitive, as compared to the HepaRG cells. The incubation with FBS-free media produced aggregation of AuNPs while its presence greatly influenced the toxicity outcomes. The cellular uptake of AuNPs was shape, size and capping dependent in PRH cultivated in FBS-supplemented media, and significantly different between the two types of cells with extensively higher internalization of AuNPs in PRH, as compared to the HepaRG cells.These data show that the physical-chemical properties of AuNPs, including size and shape, as well as the type of cellular model, greatly influence the interaction of the AuNPs with the biological environment and consequently, their toxicological effects.     

A genomic characterization of the influence of silver nanoparticles on bone differentiation in MC3T3‐E1 cells

Silver nanoparticles (AgNPs) have been widely used in a variety of biomedical applications. Previous studies demonstrated that AgNPs significantly enhanced bone cell mineralization and differentiation in MC3T3–1 cells, a model in vitro system, when compared to several other NPs. This increased bone deposition was evaluated by phenotypic measurements and assessment of the expression of miRNAs associated with regulation of bone morphogenic proteins. In the present study, we used RNA‐seq technology, a more direct measurement of gene expression, to investigate further the mechanisms of bone differentiation induced by AgNP treatment. Key factors associated with the osteoclast pathway were significantly increased in response to AgNP exposure including Bmp4, Bmp6 and Fosl1. In addition, genes of metabolism and toxicity pathways were significantly regulated as well. Although this study suggests the potential for AgNPs to influence bone morphogenesis in injury or disease applications, further investigation into the efficacy and safety of AgNPs in bone regeneration is warranted.     

Gene expression in nanotoxicology research: analysis by differential display in BALB3T3 fibroblasts exposed to cobalt particles and ions

Broadly defined, nanoscale materials are substances in which at least one critical dimension is less than 100 nm. Nanoscale materials are employed in several industrial applications as well as in biology and medicine. Despite their wide use, very little research has been carried out on the potential toxicity of nanoparticles. For this reason, we report on a molecular approach in nanotoxicology research. Using the differential display technique, we focused our attention on mRNA expression in a BALB3T3 A31-1-1 cell line that was not exposed and exposed for 72 h to 1 μM of cobalt microparticles (Co-μ), nanoparticles (Co-nano), and ions. In the experiments, we obtained 10 differentially expressed sequences. These genes represent candidate biomarkers capable of indicating specific cellular effects after Co-nano exposure. In addition, our results show that treatment with Co-nano somehow activates cellular pathways of defense and repair mechanisms. It is also evident that molecular techniques are valuable tools in nanotoxicology research, where they will certainly find wide use.     

iTRAQ: a method to elucidate cellular responses to mycotoxin zearalenone

Mycotoxin zearalenone (ZEN) is a secondary metabolite produced by some Fusarium species that contaminate a large variety of grains and feedstuffs worldwide. ZEN has been associated with a wide variety of adverse health effects including hepatotoxic, hematologic, immunotoxic and genotoxic. In order to better understand the mechanism of ZEN toxicity, a proteomic approach was applied to characterize cellular responses of hepatocarcinoma cells (HepG2) to ZEN exposure. Protein extracts from cultured HepG2 cells treated with 100 µm ZEN for 8 h, as well as extracts from control cells. The screening method applied to compare the proteome was based on the stable isotope approach of isobaric tagging for relative and absolute quantification (iTRAQ). This study identified 982 proteins, among which peptides and their corresponding proteins were identified and quantified by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. Ingenuity pathways analysis software was then used to determine the biological functions and canonical pathways associated with the ZEN‐responsive proteins. Copyright © 2012 John Wiley & Sons, Ltd.     

Oxidative stress pathways involved in cytotoxicity and genotoxicity of titanium dioxide (TiO2) nanoparticles on cells constitutive of alveolo-capillary barrier in vitro

The health risks of nanoparticles remain a serious concern given their prevalence from industrial and domestic use. The primary route of titanium dioxide nanoparticle exposure is inhalation. The extent to which nanoparticles contribute to cellular toxicity is known to associate induction of oxidative stress. To investigate this problem further, the effect of titanium dioxide nanoparticles was examined on cell lines representative of alveolo-capillary barrier.The present study showed that all nanoparticle-exposed cell lines displayed ROS generation. Macrophage-like THP-1 and HPMEC-ST1.6R microvascular cells were sensitive to endogenous redox changes and underwent apoptosis, but not alveolar epithelial A549 cells. Genotoxic potential of titanium dioxide nanoparticles was investigated using the activation of γH2AX, activation of DNA repair proteins and cell cycle arrest. In the sensitive cell lines, DNA damage was persistent and activation of DNA repair pathways was observed. Moreover, western blot analysis showed that specific pathways associated with cellular stress response were activated concomitantly with DNA repair or apoptosis.Nanoparticles-induced oxidative stress is finally signal transducer for further physiological effects including genotoxicity and cytotoxicity. Within activated pathways, HSP27 and SAPK/JNK proteins appeared as potential biomarkers of intracellular stress and of sensitivity to endogenous redox changes, respectively, enabling to predict cell behavior.     

Oxidative damage to biological macromolecules in human bone marrow mesenchymal stromal cells labeled with various types of iron oxide nanoparticles

The biological effects of several superparamagnetic iron oxide nanoparticles (SPIONs) varying in their surface coating were tested using human bone marrow mesenchymal stromal cells from two donors - hBMSCs-1 and hBMSCs-2. The measurements were performed at two intervals - after 72 h exposure to the nanoparticles and after an additional 72 h cell growth without nanoparticles. The dose of SPIONs used (15.4 μg Fe/ml) was selected as being sufficient for in vivo cell tracking using magnetic resonance imaging (MRI). Concerning cell viability and cell death, only the hBMSCs-2 seemed to be sensitive to the action of SPIONs. However, an increase of oxidative injury to lipids, proteins and DNA as a consequence of exposure to SPIONs was detected in cells from both donors. Particularly the levels of lipid peroxidation were high and increased further with time, regardless of the type of nanoparticle. Lowering intracellular label concentrations and authenticating oxidative stress levels using in vivo experiments are required to ensure the safety of SPIONs for biomedical applications.     

Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes

The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.     

In vitro cytotoxicity of superparamagnetic iron oxide nanoparticles on neuronal and glial cells. Evaluation of nanoparticle interference with viability tests

Superparamagnetic iron oxide nanoparticles (ION) have attracted great interest for use in several biomedical fields. In general, they are considered biocompatible, but little is known of their effects on the human nervous system. The main objective of this work was to evaluate the cytotoxicity of two ION (magnetite), coated with silica and oleic acid, previously determining the possible interference of the ION with the methodological procedures to assure the reliability of the results obtained. Human neuroblastoma SHSY5Y and glioblastoma A172 cells were exposed to different concentrations of ION (5–300 µg ml^–1), prepared in complete and serum‐free cell culture medium for three exposure times (3, 6 and 24 h). Cytotoxicity was evaluated by means of the MTT, neutral red uptake and alamar blue assays. Characterization of the main physical–chemical properties of the ION tested was also performed. Results demonstrated that both ION could significantly alter absorbance readings. To reduce these interferences, protocols were modified by introducing additional washing steps and cell‐free systems. Significant decreases in cell viability were observed for both cell lines in specific conditions by all assays. In general, oleic acid‐coated ION were less cytotoxic than silica‐coated ION; besides, a serum‐protective effect was observed for both ION studied and cell lines. These results contribute to increase the knowledge of the potential harmful effects of ION on the human nervous system. Understanding these effects is essential to establish satisfactory regulatory policies on the safe use of magnetite nanoparticles in biomedical applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Does photocatalytic activity of TiO2 nanoparticles correspond to photo-cytotoxicity? Cellular uptake of TiO2 nanoparticles is important in their photo-cytotoxicity

Titanium dioxide (TiO₂) nanoparticles are important industrial nano-objects with wide applications, including as photocatalysts and sunscreen components. Recently, the phototoxicity of TiO₂ nanoparticles has been a concern. However, phototoxicity caused by photocatalytic activity may differ between anatase and rutile nanoparticles. In the present study, we compared the phototoxicity of anatase and rutile nanoparticles. Human keratinocyte HaCaT cells were treated with stable TiO₂ nanoparticle suspensions. Without UVA irradiation, TiO₂ nanoparticles did not affect mitochondrial activity or cell membranes. However, exposure to rutile nanoparticle suspensions inhibited cell growth and induced HO-1 gene expression without UVA irradiation. These effects may be explained by the hydrophobic surface of rutile nanoparticles. Next, TiO₂-exposed cells were irradiated with UVA for 4?h and effects of TiO₂ nanoparticles on cells were examined. The rutile nanoparticles did not show any cellular effects after UVA irradiation. However, the anatase nanoparticles caused strong phototoxicity. Decreased mitochondrial activity, cell membrane damage and the induction of oxidative stress were observed in the cells exposed to anatase nanoparticles with UVA irradiation. Cellular uptake of the nanoparticles was observed in both anatase- and rutile-exposed cells. These results suggest that internalized anatase nanoparticles are important for phototoxicity. Additionally, the exposure of a 3D skin model to TiO₂ nanoparticles did not result in significant toxicity. In conclusion, rutile nanoparticles used in sunscreen did not exhibit phototoxic activity. Despite the strong phototoxic activity of anatase nanoparticles in cell cultures, they demonstrated no phototoxicity using a 3D skin model.     

Gold nanoparticles cellular toxicity and recovery: Adipose Derived Stromal cells

Gold nanoparticles (AuNPs) are currently used in numerous medical applications. Herein, we describe their in vitro impact on human adipose-derived stromal cells (ADSCs) using 13 nm and 45 nm citrate-coated AuNPs. In their non-differentiated state, ADSCs were penetrated by the AuNPs and stored in vacuoles. The presence of the AuNPs in ADSCs resulted in increased population doubling times, decreased cell motility and cell-mediated collagen contraction. The degree to which the cells were impacted was a function of particle concentration, where the smaller particles required a sevenfold higher concentration to have the same effect as the larger ones. Furthermore, AuNPs reduced adipogenesis as measured by lipid droplet accumulation and adiponectin secretion. These effects correlated with transient increases in DLK1 and with relative reductions in fibronectin. Upon removal of exogenous AuNPs, cellular NP levels decreased and normal ADSC functions were restored. As adiponectin helps regulate energy metabolism, local fluctuations triggered by AuNPs can lead to systemic changes. Hence, careful choice of size, concentration and clinical application duration of AuNPs is warranted.     

Uptake of silica nanoparticles: Neurotoxicity and Alzheimer-like pathology in human SK-N-SH and mouse neuro2a neuroblastoma cells

Growing concern has been raised over the potential adverse effects of engineered nanoparticles on human health due to their increasing use in commercial and medical applications. Silica nanoparticles (SiNPs) are one of the most widely used nanoparticles in industry and have been formulated for cellular and non-viral gene delivery in the central nerve system. However, the potential neurotoxicity of SiNPs remains largely unclear. In this study, we investigated the cellular uptake of SiNPs in human SK-N-SH and mouse neuro2a (N2a) neuroblastoma cells treated with 10.0 μg/ml of 15-nm SiNPs for 24 h by transmission electron microscopy. We found that SiNPs were mainly localized in the cytoplasm of the treated cells. The treatment of SiNPs at various concentrations impaired the morphology of SK-N-SH and N2a cells, characterized by increased number of round cells, diminishing of dendrite-like processes and decreased cell density. SiNPs significantly decreased the cell viability, induced cellular apoptosis, and elevated the levels of intracellular reactive oxygen species (ROS) in a dose-dependent manner in both cell lines. Additionally, increased deposit of intracellular β-amyloid 1-42 (Aβ_1-42) and enhanced phosphorylation of tau at Ser262 and Ser396, two specific pathological hallmarks of Alzheimer's disease (AD), were observed in both cell lines with SiNPs treatment. Concomitantly, the expression of amyloid precursor protein (APP) was up-regulated, while amyloid-β-degrading enzyme neprilysin was down-regulated in SiNP-treated cells. Finally, activity-dependent phosphorylation of glycogen syntheses kinase (GSK)-3β at Ser9 (inactive form) was significantly decreased in SiNP-treated SK-N-SH cells. Taken together, these data demonstrated that exposure to SiNPs induced neurotoxicity and pathological signs of AD. The pre-Alzheimer-like pathology induced by SiNPs might result from the dys-regulated expression of APP/neprilysin and activation of GSK-3β. This is the first study with direct evidence indicating that in addition to neurotoxicity induced by SiNPs, the application of SiNPs might increase the risk of developing AD.