BackgroundNanomaterials can be contaminated with endotoxin (lipopolysaccharides, LPS) during production or handling. In this study, we searched for a convenient in vitro method to evaluate endotoxin contamination in nanoparticle samples. We assessed the reliability of the commonly used limulus amebocyte lysate (LAL) assay and an alternative method based on toll-like receptor (TLR) 4 reporter cells when applied with particles (TiO2, Ag, CaCO3 and SiO2), or after extraction of the endotoxin as described in the ISO norm 29701.ResultsOur results indicate that the gel clot LAL assay is easily disturbed in the presence of nanoparticles; and that the endotoxin extraction protocol is not suitable at high particle concentrations. The chromogenic-based LAL endotoxin detection systems (chromogenic LAL assay and Endosafe-PTS), and the TLR4 reporter cells were not significantly perturbed.ConclusionWe demonstrated that nanoparticles can interfere with endotoxin detection systems indicating that a convenient test method must be chosen before assessing endotoxin contamination in nanoparticle samples.

The unique physical and chemical properties of nanomaterials have led to their increased use in many industrial applications, including as a paint additive. For example, titanium dioxide (TiO2) engineered nanoparticles (ENPs) have well-established anti-UV, self-cleaning, and air purification effects. Silver (Ag) ENPs are renowned for their anti-microbial capabilities and silicon dioxide (SiO2) ENPs are used as fire retardants and anti-scratch coatings. In this study, the toxic effects and biodistribution of three pristine ENPs (TiO2, Ag, and SiO2), three aged paints containing ENPs (TiO2, Ag, and SiO2) along with control paints without ENPs were compared. BALB/c mice were oropharyngeally aspirated with ENPs or paint particles (20 μg/aspiration) once a week for 5 weeks and sacrificed either 2 or 28 days post final aspiration treatment. A bronchoalveolar lavage was performed and systemic blood toxicity was evaluated to ascertain cell counts, induction of inflammatory cytokines, and key blood parameters. In addition, the lung, liver, kidney, spleen, and heart were harvested and metal concentrations were determined. Exposure to pristine ENPs caused subtle effects in the lungs and negligible alterations in the blood. The most pronounced toxic effects were observed after Ag ENPs exposure; an increased neutrophil count and a twofold increase in pro-inflammatory cytokine secretion (keratinocyte chemoattractant (KC) and interleukin-1ß (IL-1ß)) were identified. The paint containing TiO2 ENPs did not modify macrophage and neutrophil counts, but mildly induced KC and IL-1ß. The paints containing Ag or SiO2 did not show significant toxicity. Biodistribution experiments showed distribution of Ag and Si outside the lung after aspiration to respectively pristine Ag or SiO2 ENPs. In conclusion, we demonstrated that even though direct exposure to ENPs induced some toxic effects, once they were embedded in a complex paint matrix little to no adverse toxicological effects were identified.

We determined the ability of a model nanoparticle (NP) (titanium dioxide, TiO2) to modulate sensitization induced by a known potent dermal sensitizer (dinitrochlorobenzene) using a variant of the local lymph node assay called lymph node proliferation assay.BALB/c mice received sub-cutaneous injections of vehicle (2.5 mM sodium citrate), TiO2 NPs (0.004, 0.04 or 0.4 mg/ml) or pigment particles (0.04 mg/ml) both stabilized in sodium citrate buffer at the base of each ear (2x50μl), before receiving dermal applications (on both ears) of 2,4-Dinitrochlorobenzene (DNCB) (2x25μl of 0.1%) or its vehicle (acetone olive oil – AOO (4:1)) on days 0, 1 and 2. On day 5, the stimulation index (SI) was calculated as a ratio of 3HTdR incorporation in lymphocytes from DNBC-treated mice and AOO-treated controls. In a second experiment the EC3-value for DNCB (0 to 0.1%) was assessed in the absence or presence of 0.04 mg/ml TiO2. In a third experiment, the lymphocyte subpopulations and the cytokine secretion profile were analyzed after TiO2 (0.04 mg/ml) and DNCB (0.1%) treatment.Injection of NPs in AOO-treated control mice did not have any effect on lymph node (LN) proliferation. DNCB sensitization resulted in LN proliferation, which was further increased by injection of TiO2 NPs before DNCB sensitization. The EC3 of DNCB, with prior injection of vehicle control was 0.041%, while injection with TiO2 decreased the EC3 of DNCB to 0.015%. TiO2 NPs pre-treatment did not alter the lymphocyte subpopulations, but significantly increased the level of IL-4 and decreased IL-10 production in DNCB treated animals.In conclusion, our study demonstrates that administration of nano-TiO2 increases the dermal sensitization potency of DNCB, by augmenting a Th2 response, showing the immunomodulatory abilities of NPs.

We have demonstrated that topical exposure to TiO2 ENPs increases chemical-induced dermal sensitization.

Large knowledge gaps still exist on the toxicological mechanisms of silver (Ag) engineered nanoparticles (ENPs); a comprehensive understanding of the sources, biodistribution, toxicity and transformation of Ag ENPs along their life cycle and after transfer in living organisms is needed. In a previous study, mice were pulmonary exposed to Ag ENPs and local (lung) and systemic toxic effects together with biodistribution to organs including heart, liver, spleen and kidney were investigated. Here, Ag lung distribution, local concentration, co-localization with other elements such as Fe, Cu and S, and speciation were determined after lung exposure to Ag ENPs using micro X-ray fluorescence (μXRF), micro X-ray absorption near edge structure spectroscopy (μXANES) and micro proton-induced X-ray emission (μPIXE) techniques. We found that approximately a quarter of all macrophages in the lumen of the airways contained ENPs. High local concentrations of Ag were also detected in the lung tissue, probably phagocytized by macrophages. The largest part of the ENPs was dissolved and complexed to thiol-containing molecules. Increased concentrations of Fe and Cu observed in the Ag-rich spots suggest that these molecules are metallothioneins (MTs). These results give more insights on the behavior of Ag ENPs in the lung in vivo and will help in the understanding of the toxicological mechanisms of Ag ENPs.