Christelle Saout

Copper(II) oxide nanoparticles penetrate into HepG2 cells, exert cytotoxicity via oxidative stress and induce pro-inflammatory response

The potential toxic effects of two types of copper(II) oxide (CuO) nanoparticles (NPs) with different specific surface areas, different shapes (rod or spheric), different sizes as raw materials and similar hydrodynamic diameter in suspension were studied on human hepatocarcinoma HepG2 cells. Both CuO NPs were shown to be able to enter into HepG2 cells and induce cellular toxicity by generating reactive oxygen species. CuO NPs increased the abundance of several transcripts coding for pro-inflammatory interleukins and chemokines. Transcriptomic data, siRNA knockdown and DNA binding activities suggested that Nrf2, NF-κB and AP-1 were implicated in the response of HepG2 cells to CuO NPs. CuO NP incubation also induced activation of MAPK pathways, ERKs and JNK/SAPK, playing a major role in the activation of AP-1. In addition, cytotoxicity, inflammatory and antioxidative responses and activation of intracellular transduction pathways induced by rod-shaped CuO NPs were more important than spherical CuO NPs. Measurement of Cu(2+) released in cell culture medium suggested that Cu(2+) cations released from CuO NPs were involved only to a small extent in the toxicity induced by these NPs on HepG2 cells.

Eco-, geno- and human toxicology of bio-active nanoparticles for biomedical applications

Gene delivery has become an increasingly important strategy for treating a variety of human diseases, including infections, genetic disorders and tumours. To avoid the difficulties of using viral carriers, more and more non-viral gene delivery nanoparticles are developed. Among these new approaches polyethylene imine (PEI) is currently considered as one of the most effective polymer based method solution and considered as the gold standard. The toxicity of nanoparticles is a major concern when used for medical application. In this study we chose two nanoparticles for an in depth toxicological and ecotoxicological evaluation, one well characterized, PEI, and another novel polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). In the present study we have assessed the toxicity of these cation nanoparticles as such and of the polyplexes - nanoparticles covered with DNA. As these nanoparticles are also frequently used in high volumes in various industries and as such may enter in the environment, we also made an initial assessment of ecotoxicological effects assessment. The following nanoparticles related aspects have been studied during the project: development and characterization, ecotoxicity, general toxicity and specific toxicity. To this end a battery of different tests was used. The conclusion of these tests is that toxicity is varying between different nanoparticles and between different DNA covering ratios. In general, in the different systems tested, the PEI polymer is more toxic than the PDMAEMA polymer. The same difference is seen for the polyplexes and the higher the charge ratio, the more toxic are the polyplexes. Our study also clearly shows the need for a broad spectrum of toxicity assays for a comprehensive risk assessment. Our study has performed such a comprehensive analysis of two biomedical nanoparticles.

Differential toxicity of copper (II) oxide nanoparticles of similar hydrodynamic diameter on human differentiated intestinal Caco-2 cell monolayers is correlated in part to copper release and shape

Abstract The potential toxic effects of copper oxide (CuO) nanoparticles (NPs) were studied on differentiated Caco-2 cell monolayers, a classical in vitro model of human small intestine epithelium. Two types of CuO NPs, with different specific surface area, different sizes as raw material but the same hydrodynamic diameter in suspension, differentially disturbed the monolayer integrity, were cytotoxic and triggered an increase of the abundance of several transcripts coding for pro-inflammatory cytokines and chemokines. Specific surface area was not a major variable explaining the increased toxicity when intestinal epithelium is exposed to rod-shaped CuO NPs, compared with spherical CuO NPs. The results suggest that release of Cu(II) cations and shape of these CuO NPs are likely to be implicated in the toxicity of these CuO NPs.

Cytotoxicity of multi-walled carbon nanotubes in three skin cellular models: Effects of sonication, dispersive agents and corneous layer of reconstructed epidermis

Abstract The effects of multi-walled carbon nanotubes were investigated in SZ95 sebocytes, IHK keratinocytes and reconstructed human epidermises. Carbon nanotubes were subjected to dispersion protocols leading to different agglomeration states. Toxicological methods were chosen and adapted in order to ensure compatibility with nanotubes. Results show that: (i) Water-suspended nanotubes, as micrometric agglomerates, were not harmful to skin cells, except minor effects in keratinocytes, (ii) mild sonication slightly decreased nanotube agglomeration but increased cytotoxicity on keratinocytes, (iii) addition of hydroxypropylcellulose or Pluronic F108, which improved nanotube dispersion, masked the harmful effects of sonicated nanotubes. Altogether, these results indicate that carbon nanotubes induced cytotoxicity in human keratinocytes after a short exposure (24–48 h), particularly when they were sonicated before cell incubations. However, the cytotoxic effects of raw and sonicated nanotubes could be prevented in presence of dispersive agents. No cytotoxic effects were observed in SZ95 sebocytes or in stratified epidermises reconstructed in vitro.

How does the deposited dose of oxide nanomaterials evolve in an in vitro assay

In this work, the evolution of some key physicochemical parameters of oxide engineered nanomaterial (ENM) dispersions was studied during an in vitro biological assessment. Commercial oxide ENMs, SiO2 and TiO2, were dispersed in aqueous solutions (20 μg/mL) to A549 cells and N-hTERT keratinocytes and were assessed at several incubation times: 6, 24, 48, and 72 hours. The ENMs deposited dose and its particle size distribution (PSD) were followed each time. Centrifuge Liquid Sedimentation (CLS) measured the PSD and the ENMs deposited dose from a particle entity perspective, while Particle-Induced X-ray Emission (PIXE) measured the ENMs deposited dose from an elemental mass perspective. No significant variations in PSD were observed for SiO2 ENMs during incubation in A549 cells and TiO2 ENMs in both cell lines, while a continuous evolution of the PSD is observed for SiO2 in N-hTERT keratinocytes. The deposited dose for TiO2 ENMs remained stable and similar in both cell lines due to a smaller specific surface area and a higher quantity of primary particles present during incubation. It is concluded that the observed differences in the deposited dose are related to an interaction between the proteins present in the media and the ENMs specific surface.

Pro-inflammatory effects of different MWCNTs dispersions in p16INK4A-deficient telomerase-expressing human keratinocytes but not in human SV-40 immortalized sebocytes

Abstract We tested whether multi-walled carbon nanotubes (MWCNTs) induce oxidative stress and a pro-inflammatory response in human N-hTERT telomerase-immortalized keratinocytes, in human SZ95 SV-40 immortalized sebocytes and in in vitro reconstructed epidermises. MWCNTS were tested in various dispersion states, from raw and agglomerated particles to isolated entities obtained by sonication in the presence of dispersive agents (hydroxypropylcellulose and Pluronic F108). It was observed that: (a) Contrary to individualized MWCNTs, agglomerated particles prepared by suspension into pure water increased the intracellular levels of reactive oxygen species as well as the expression and secretion of interleukin-8 in N-hTERT cells; (b) the inflammatory signature of MWCNTs in N-hTERT cells, drawn by transcriptomic analysis with low-density microfluidic cards, included various other cytokines such as interleukin-6 or C-C motif ligand 3; (c) the pro-inflammatory effects of MWCNTs, as assessed by interleukin-8 transcript level and protein release, were not observed in SZ95 cells; and (d) the secretion of interleukins-1α and -8 from in vitro reconstructed epidermal tissues, used as specific markers for skin irritation and sensitization, was unaffected in presence of MWCNTs, confirming that the cornified layer is an efficient barrier against MWCNTs.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-β and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-β and TNF-α measurements.

Inflammation response at the transcriptional level of HepG2 cells induced by multi-walled carbon nanotubes

Poor information are currently available about the biological effects of multi-walled carbon nanotubes (MWCNT) on the liver. In this study, we evaluated the effects of MWCNT at the transcriptional level on the classical in vitro model of HepG2 hepatocarcinoma cells. The expression levels of 96 transcript species implicated in the inflammatory and immune responses was studied after a 24h incubation of HepG2 cells in presence of raw MWCNT dispersed in water by stirring. Among the 46 transcript species detected, only a few transcripts including mRNA coding for interleukine-7, chemokines receptor of the C-C families CCR7, as well as Endothelin-1, were statistically more abundant after treatment with MWCNT. Altogether, these data indicate that MWCNT can only induce a weak inflammatory response in HepG2 cells.