Assay conditions can influence the outcome of cytotoxicity tests of nanomaterials: better assay characterization is needed to compare studies.

BACKGROUND: Reliable in vitro studies that generate consistent toxicity data on nanomaterials on a high-throughput scale will be of invaluable significance in the next few years. AIM: In this study, we checked the influence of several steps of the experimental design on the outcome: we investigated the role of cell density, viability assay and particle dispersion method, including the influence of serum and effect of a surfactant (Tween 80). METHODS: The dose-response curve was assessed for ground multiwalled carbon nanotubes (CNT) and the silica benchmark Min-U-Sil, on lung epithelial cells (A549 cells) and macrophages (stimulated THP-1 cells). RESULTS: The cell density used in the cytotoxicity study has an impact on the outcome: for the same concentration of Min-U-Sil, the viability of A549 cells varied from 10% to 55% with increasing cell density. Whereas foetal calf serum attenuated the cytotoxicity of Min-U-Sil, this effect was not seen for CNT. The results show how cell culture conditions can modify the outcome of a toxicological experiment, as shown in this study for Tween 80 to disperse the test agent. CONCLUSIONS: These experiments illustrates that results reported in literature can only be compared when, in addition to the use of a benchmark particle, a detailed method description is available. Therefore, more emphasis is needed on a standardized design for cytotoxicity studies.

In vitro translocation of quantum dots and influence of oxidative stress.

In vivo, translocation of inhaled nanoparticles to the circulation has been demonstrated. However, the interaction of nanoparticles with the lung epithelium is not understood. In this study, we investigated, in vitro, the translocation of nano-sized quantum dots (QDs; 25 pmol/ml) through a tight monolayer of primary isolated rat alveolar epithelial cells. The influence of surface charge on translocation was examined using nonfunctionalized QDs, amine-QDs, and carboxyl-QDs. The interaction between nanoparticles and the lung epithelium was monitored by repeatedly measuring the transepithelial electrical resistance (TEER) and by examining the cell layer with confocal microscopy. The effect of oxidative stress was tested by incubating the cells with tert-butyl hydroperoxide (t-BOOH; 75 microM or 1 or 10 mM); the antioxidant N-acetyl-L-cysteine was also used to assess the role of particle-mediated oxidative stress. No translocation through a tight monolayer of primary rat alveolar epithelial cells was observed for any of the different types of QDs. In general, an increase in TEER was found after incubation with QDs. A condition of low oxidative stress did not enhance translocation. In contrast, conditions of high stress (1 or 10 mM t-BOOH or due to QDs toxicity) with disruption of the cell layer, as shown in a decreased TEER, resulted in substantial translocation. In conclusion, no translocation of QDs was found through a tight monolayer of primary rat alveolar epithelial cells, regardless of the QDs surface charge. QDs did not impair the barrier function of the epithelial cells. In conditions with disruption of the cell-cell barrier, translocation was demonstrated.

Do nanomedicines require novel safety assessments to ensure their safety for long-term human use?

Nanomaterials have different chemical, physical and biological characteristics than larger materials of the same chemical composition. These differences give nanotechnology a double identity: their use implies novel and interesting medical and/or industrial applications but also potential danger for human and environmental health. Here, we briefly review the most important types of nanomaterials, the difficulties in assessing safety or toxicity, and describe existing test protocols used in nanomaterial safety evaluation. In general, the big challenge of nanotechnology, particularly for nanomedicine (nano-bioengineering), is to understand which nano-specific characteristics interact with particular biological systems and functions in order to optimize the therapeutic potential and reduce the undesired responses. The evaluation of the safety of medicinal nanomaterials, especially for long-term application, is an important challenge for the near future. At present, it is still too early to predict, on the basis of the characteristics of the nanomaterial, a possible biological response because no reliable database exists. Therefore, a case-by-case approach for hazard identification is still required, so it is difficult to establish a risk assessment framework.

Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines.

BACKGROUND: A critical issue with nanomaterials is the clear understanding of their potential toxicity. We evaluated the toxic effect of 24 nanoparticles of similar equivalent spherical diameter and various elemental compositions on 2 human pulmonary cell lines: A549 and THP-1. A secondary aim was to elaborate a generic experimental set-up that would allow the rapid screening of cytotoxic effect of nanoparticles. We therefore compared 2 cytotoxicity assays (MTT and Neutral Red) and analyzed 2 time points (3 and 24 hours) for each cell type and nanoparticle. When possible, TC50 (Toxic Concentration 50 i.e. nanoparticle concentration inducing 50% cell mortality) was calculated. RESULTS: The use of MTT assay on THP-1 cells exposed for 24 hours appears to be the most sensitive experimental design to assess the cytotoxic effect of one nanoparticle. With this experimental set-up, Copper- and Zinc-based nanoparticles appear to be the most toxic. Titania, Alumina, Ceria and Zirconia-based nanoparticles show moderate toxicity, and no toxicity was observed for Tungsten Carbide. No correlation between cytotoxicity and equivalent spherical diameter or specific surface area was found. CONCLUSION: Our study clearly highlights the difference of sensitivity between cell types and cytotoxicity assays that has to be carefully taken into account when assessing nanoparticles toxicity.

Acute toxicity and prothrombotic effects of quantum dots: impact of surface charge.

BACKGROUND: Quantum dots (QDs) have numerous possible applications for in vivo imaging. However, toxicity data are scarce. OBJECTIVES: To determine the acute in vivo toxicity of QDs with carboxyl surface coating (carboxyl-QDs) and QDs with amine surface coating (amine-QDs), we investigated the inflammatory properties, tissue distribution, and prothrombotic effects after intravenous injection. METHODS: We performed particle characterization by transmission electron microscopy and dynamic light scattering. Carboxyl-QDs and amine-QDs were intravenously injected in mice (1.44-3,600 pmol/mouse). At different time intervals, analyses included fluorescence microscopy, blood cell analysis, bronchoalveolar lavage, wet and dry organ weights, and cadmium concentration in various organs. We examined the prothrombotic effects in vivo by assessing the effect of pretreatment with the anticoagulant heparin and by measuring platelet activation (P-selectin), and in vitro by platelet aggregation in murine and human platelet-rich plasma exposed to QDs (1.44-1,620 pmol/mL). RESULTS: At doses of 3,600 and 720 pmol/mouse, QDs caused marked vascular thrombosis in the pulmonary circulation, especially with carboxyl-QDs. We saw an effect of surface charge for all the parameters tested. QDs were mainly found in lung, liver, and blood. Thrombotic complications were abolished, and P-selectin was not affected by pretreatment of the animals with heparin. In vitro, carboxyl-QDs and amine-QDs enhanced adenosine-5'-diphosphate-induced platelet aggregation. CONCLUSION: At high doses, QDs caused pulmonary vascular thrombosis, most likely by activating the coagulation cascade via contact activation. Our study highlights the need for careful safety evaluation of QDs before their use in human applications. Furthermore, it is clear that surface charge is an important parameter in nanotoxicity.

Immunization with the binding domain of FimH, the adhesin of type 1 fimbriae, does not protect chickens against avian pathogenic Escherichia coli.

The aim of this study was to investigate whether vaccination with the sugar-binding domain of FimH (FimH156) was able to protect chickens against avian pathogenic Escherichia coli (APEC). FimH156 was expressed and purified using Ni-NTA affinity chromatography. Binding of FimH156 to mannosylated bovine serum albumin demonstrated that the protein retained its biological activity. Moreover, anti-FimH156 antisera were able to inhibit in vitro binding of E. coli to mannosylated bovine serum albumin. In a first vaccination experiment, FimH156 was administered intramuscularly as a water-in-oil emulsion to specific pathogen free broiler chicks. A predisposing infection with the Newcastle disease virus strain Lasota was administered 3 weeks later, followed 3 days later by an aerosol challenge with the virulent APEC strain CH2. A good anti-FimH156 immunoglobulin (Ig)G immune response was detected in serum, but no protective effects of FimH156 against APEC were seen. In a second experiment, SPF chicks were vaccinated intramuscularly or intranasally with FimH156. Booster vaccinations were administered 20 days later. While the intramuscular immunization yielded a strong IgG response in the serum and trachea, no significant IgA response could be detected in tracheal washes. Intranasal immunization did not yield a significant IgG or IgA response in serum and trachea. No protective effects of the FimH156 could be detected, confirming the results of the first experiment. Thus, although the FimH156 induced a strong immune response, it was unable to protect chickens against APEC.