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Toxicity determinants of multi-walled carbon nanotubes: The relationship between functionalization and agglomeration.

ABSTRACT: The elucidation of toxicity determinants of multi-walled carbon nanotubes (MWCNT) is still incomplete. Functionalization with carboxyl groups is, however, commonly used to mitigate MWCNT toxicity, although the rationale for the mitigating effect has not been fully clarified yet. In this work, two optimized chemical vapor deposition methods were employed to obtain MWCNT of comparable length but different diameter, which were subsequently functionalized. For MWCNT of diameter larger than 40 nm, no detrimental effects on cell viability of macrophages were observed, while mild cytotoxicity was recorded for diameters between 15 and 40 nm, with a mitigating effect of functionalization. To investigate the factors responsible for the mitigation, we used the thinnest MWCNT preparation on different cell models, evaluating several endpoints, such as viability, production of nitric oxide (NO), expression of pro-inflammatory markers, the Trans-Epithelial Electrical Resistance (TEER), and clonogenic activity. Substantial mitigation of the changes caused by pristine MWCNT was observed not only with carboxyl- but also with amino-functionalized MWCNT, suggesting that negative or positive surface charge was not the main factor responsible for the effect. Instead, either functionalized preparation exhibited a stronger tendency to agglomerate that was strictly dependent on the presence of proteins. Moreover, we found that either carboxyl- or amino-functionalized MWCNT adsorbed a larger amount of serum proteins than pristine counterparts, with a distinctive pattern for each type of MWCNT. We propose, therefore, that the formation of larger agglomerates, dependent upon different protein coronae, contributes to mitigate the biological effects of functionalized MWCNT in protein-rich biological media.

SUBMITTER: Allegri M

PROVIDER: S-EPMC5615827 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Toxicity determinants of multi-walled carbon nanotubes: The relationship between functionalization and agglomeration.

Allegri Manfredi M Perivoliotis Dimitrios K DK Bianchi Massimiliano G MG Chiu Martina M Pagliaro Alessandra A Koklioti Malamatenia A MA Trompeta Aikaterini-Flora A AA Bergamaschi Enrico E Bussolati Ovidio O Charitidis Constantinos A CA

Toxicology reports 20160119

The elucidation of toxicity determinants of multi-walled carbon nanotubes (MWCNT) is still incomplete. Functionalization with carboxyl groups is, however, commonly used to mitigate MWCNT toxicity, although the rationale for the mitigating effect has not been fully clarified yet. In this work, two optimized chemical vapor deposition methods were employed to obtain MWCNT of comparable length but different diameter, which were subsequently functionalized. For MWCNT of diameter larger than 40 nm, no ...[more]

PMID: 28959543

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Project description:BackgroundThe wide application of multi-walled carbon nanotubes (MWCNTs) in various fields has raised enormous concerns regarding their safety for humans. However, studies on the toxicity of MWCNTs to the eye are rare and potential molecular mechanisms are completely lacking. This study was to evaluate the adverse effects and toxic mechanisms of MWCNTs on human ocular cells.MethodsHuman retinal pigment epithelial cells (ARPE-19) were treated with pristine MWCNTs (7-11 nm) (0, 25, 50, 100 or 200 μg/mL) for 24 hours. MWCNTs uptake into ARPE-19 cells was examined using transmission electron microscopy (TEM). The cytotoxicity was evaluated by CCK-8 assay. The death cells were detected by Annexin V-FITC/PI assay. RNA profiles in MWCNT-exposed and non-exposed cells (n = 3) were analyzed using RNA-sequencing. The differentially expressed genes (DEGs) were identified through the DESeq2 method and hub of which were filtered by weighted gene co-expression, protein-protein interaction (PPI) and lncRNA-mRNA co-expression network analyses. The mRNA and protein expression levels of crucial genes were verified using quantitative polymerase chain reaction (qPCR), colorimetric analysis, ELISA and Western blotting. The toxicity and mechanisms of MWCNTs were also validated in human corneal epithelial cells (HCE-T).ResultsTEM analysis indicated the internalization of MWCNTs into ARPE-19 cells to cause cell damage. Compared with untreated ARPE-19 cells, those exposed to MWCNTs exhibited significantly decreased cell viabilities in a dose-dependent manner. The percentages of apoptotic (early, Annexin V positive; late, Annexin V and PI positive) and necrotic (PI positive) cells were significantly increased after exposure to IC50 concentration (100 μg/mL). A total of 703 genes were identified as DEGs; 254 and 56 of them were, respectively, included in darkorange2 and brown1 modules that were significantly associated with MWCNT exposure. Inflammation-related genes (including CXCL8, MMP1, CASP3, FOS, CXCL2 and IL11) were screened as hub genes by calculating the topological characteristics of genes in the PPI network. Two dysregulated long non-coding RNAs (LUCAT1 and SCAT8) were shown to regulate these inflammation-related genes in the co-expression network. The mRNA levels of all eight genes were confirmed to be upregulated, while caspase-3 activity and the release of CXCL8, MMP1, CXCL2, IL11 and FOS proteins were demonstrated to be increased in MWCNT-treated ARPE-19 cells. MWCNTs exposure also can induce cytotoxicity and increase the caspase-3 activity and the expression of LUCAT1, MMP1, CXCL2, and IL11 mRNA and protein in HCE-T cells.ConclusionOur study provides promising biomarkers for monitoring MWCNT-induced eye disorders and targets for developing preventive and therapeutic strategies.

Dataset Information

Toxicity determinants of multi-walled carbon nanotubes: The relationship between functionalization and agglomeration.

Publications

Toxicity determinants of multi-walled carbon nanotubes: The relationship between functionalization and agglomeration.

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