An in vitro strategy to assess mitigation of hazardous properties of engineered metal nanoparticles

The huge progress in the nanotechnology field has requested the production of increasingly advanced engineered nanoparticles (NPs). In particular, metal-based advanced NPs are widely used in several industrial applications. However, their potential effects on human health during occupational exposure are still incompletely characterized thus far and possible strategies to decrease their hazardous properties are not yet clearly defined. In this project we are developing an in vitro approach to test the cytotoxic effects of metal-based NPs, as derived from production lines or modified through coating with organic or inorganic moieties. We have used two cell models widely employed in toxicological studies, the human alveolar cell line A549 and the murine macrophage cell line RAW264.7, to avoid possible limitations due to cell specific effects. Moreover, in order to evaluate the effectiveness of mitigation approaches for NPs endowed with little acute cytotoxicity, additional endpoints, alternative to viability, have also been assessed. Colloidal suspensions of Ag, TiO2 and ZrO2 NPs were tested as provided by industries or modified with SiO2 NPs or citrate used as coating remediation agents. Heterocoagulation of opposite charged phases was applied in order to promote the coating of pristine surfaces by modifying agents. Heterocoagulated sols were obtained by ball milling sols of positive charged Ag, TiO2 and ZrO2 NPs with negative charged SiO2 NPs or citrate ions. Modified samples, obtained by spray-drying and re- dispersing in water the corresponding sols, were also obtained in order to compare reactivity. Original and modified NPs were added to culture media starting from water colloidal suspensions. Viability was determined with the resazurin method in a range of doses from 2.5 to 80 nfg/cm2 (0.3125 to 20 g/cm2 for Ag NPs) of monolayer surface at three experimental times (24, 48 and 72h). The expression of the inducible form of nitric oxide synthase (Nos2), an indicator of macrophage activation and, hence, of pro-inflammatory activity, was assessed with RT-PCR as an end-point alternative to viability. Among the NPs tested, only Ag NP caused a significant loss of viability, with an IC50 of about 0.8 g/cm2 for Raw264.7 cells and 2.4 g/cm2 for A549 cells at the 24h-experimental time. In a preliminary experiment, SiO2 NPs were demonstrated to have no significant effect on cell viability. The comparison between original and SiO2-coated Ag NPs, performed in the same experiment, suggested a coating-independent mitigation effect of bioreactivity exerted by the spray drying procedure. However, once corrected for the actual Ag content of the spray- dried powder, no significant difference was found in the IC50 values, indicating that neither silica coating nor spray drying mitigate cytotoxicity. The effects on viability of original TiO2 and ZrO2 NPs were assessed using P25 Aeroxide TiO2 NPs as a reference material. These materials did not affect significantly cell viability at any time point tested, so that it was not possible to estimate IC50 values for either cell line. However, titania produced a clear-cut induction of Nos2 expression in Raw264.7 cells, thus indicating their potential pro- inflammatory activity. Citrate coating did not produce any significant attenuation of the biological effect. In summary, these preliminary results showed no mitigating effect of the surface modifications tested on the biological effects of the engineered NPs investigated. However, the exploitation of this in vitro experimental strategy can be useful for the preliminary assessment of the mitigation potential of surface modifications of both low-toxic and high-toxic engineered NPs. Supported by EU Grant NMP4-SL-2012-280716 (Sanowork Project

Adherence issues related to sublingual immunotherapy as perceived by allergists

Objectives: Sublingual immunotherapy (SLIT) is a viable alternative to subcutaneous immunotherapy to treat allergic rhinitis and asthma, and is widely used in clinical practice in many European countries. The clinical efficacy of SLIT has been established in a number of clinical trials and meta-analyses. However, because SLIT is self-administered by patients without medical supervision, the degree of patient adherence with treatment is still a concern. The objective of this study was to evaluate the perception by allergists of issues related to SLIT adherence. Methods: We performed a questionnaire-based survey of 296 Italian allergists, based on the adherence issues known from previous studies. The perception of importance of each item was assessed by a VAS scale ranging from 0 to 10. Results: Patient perception of clinical efficacy was considered the most important factor (ranked 1 by 54% of allergists), followed by the possibility of reimbursement (ranked 1 by 34%), and by the absence of side effects (ranked 1 by 21%). Patient education, regular follow-up, and ease of use of SLIT were ranked first by less than 20% of allergists. Conclusion: These findings indicate that clinical efficacy, cost, and side effects are perceived as the major issues influencing patient adherence to SLIT, and that further improvement of adherence is likely to be achieved by improving the patient information provided by prescribers. © 2010 Scurati et al, publisher and licensee Dove Medical Press Ltd

TiO2 Nanoparticles synergistically increase LPS-induced NO Production by Macrophages

Although TiO2 NPs particles are endowed with little acute toxicity in vitro, they exert pro-inflammatory effects when inhaled in vivo. In this study, we evaluated the effects of several TiO2NPs, alone or in combination with LPS, on Raw264.7 macrophages in terms of cell viability (determined up to 72h with resazurin assay), NO production (estimated by nitrite concentration in the culture medium after 48h and 72h of treatment), and Nos2 induction, assessed with RT-PCR after 24h of treatment. We tested three types of TiO2 NP: a) pristine TiO2 NP of industrial origin (84% anatase/16% brookite, average NP size 45 nm), b) citrate-coated TiO2 NP, derived from pristine TiO2 NPs (average NP size 64 nm), c) Aeroxide P25 TiO2 NPs (80% anatase/20% rutile, average NP size 25 nm). All the TiO2 NPs did not affect significantly cell viability even at the highest doses tested (IC50>80 µg/cm2 at 24,48 and 72h). On the contrary, all the NP preparations, at the dose of 20 µg/cm2, induced Nos2. In particular, Nos2 was 3-fold, 8-fold and 4-fold induced by pristine, citrate coated and Aeroxide P25 TiO2 NPs alone, while fold-induction rose to 13 for pristine and citrate-coated NPs and to 30 for Aeroxide P25 NPs in the presence of 1 ng/ml LPS. LPS alone caused a 6-fold increase in Nos2 expression. The effects of pristine, citrate-coated and Aeroxide P25 TiO2 NPs on NO production were clearly dose- and time-dependent. At 80 g/cm2 of NP, nitrites were 5-fold, 9-fold and 7-fold higher than in the untreated control at 48h and 7-fold,10-fold and 8-fold higher than control at 72h. For all the TiO2NPs tested, the No-Observed-Effect-Level (NOEL) was 20 g/cm2 at 48h and 10 g/cm2 at 72h. After 48h-incubation with LPS (1 ng/ml) and TiO2 (80 µg/cm2), nitrite medium concentration was 31-fold, 30-fold and 25-fold higher than control with, respectively, pristine, citrate-coated and Aeroxide P25. LPS alone caused a 22-fold increase in medium nitrites. Several TiO2 NPs significantly induce Nos2 gene expression and increase NO production in a dose- and time-dependent manner in Raw264.7 cells. The enhancement of Nos2 induction by LPS suggests that the pro-inflammatory effect of TiO2 NPs in vivo may be exacerbated by concomitant infectious conditions and surface interactions with bacterial endotoxins. Supported by EU Grant NMP4-SL-2012-280716 (Sanowork Project

Lipopolysaccharide Adsorbed to the Bio-Corona of TiO2 Nanoparticles Powerfully Activates Selected Pro-inflammatory Transduction Pathways

It is known that the adsorption of bioactive molecules provides engineered nanoparticles (NPs) with novel biological activities. However, the biological effects of the adsorbed molecules may also be modified by the interaction with NP. Bacterial lipopolysaccharide (LPS), a powerful pro-inflammatory compound, is a common environmental contaminant and is present in several body compartments such as the gut. We recently observed that the co-incubation of LPS with TiO2 NPs markedly potentiates its pro-inflammatory effects on murine macrophages, suggesting that, when included in a NP bio-corona, LPS activity is enhanced. To distinguish the effects of adsorbed LPS from those of the free endotoxin, a pellet fraction, denominated P25/LPS, was isolated by centrifugation from a mixture of P25 TiO2 NP (128 µg/ml) and LPS (10 ng/ml) in the presence of fetal bovine serum. Western blot analysis of the pellet eluate indicated that the P25/LPS fraction contained, besides proteins, also LPS, pointing to the presence of LPS-doped NP. The effects of adsorbed or free LPS were then compared in Raw264.7 murine macrophages. RT-PCR was used to evaluate the induction of cytokine genes, whereas active, phosphorylated isoforms of proteins involved in signaling pathways were assessed with western blot. At a nominal LPS concentration of 40 pg/ml, P25/LPS induced the expression of both NF-κB and IRF3-dependent cytokines at levels comparable with those observed with free LPS (10 ng/ml), although with different time courses. Moreover, compared to free LPS, P25/LPS caused a more sustained phosphorylation of p38 MAPK and a more prolonged induction of STAT1-dependent genes. Cytochalasin B partially inhibited the induction of Tnfa by P25/LPS, but not by free LPS, and suppressed the induction of IRF3-dependent genes by either P25/LPS or free LPS. These data suggest that, when included in the bio-corona of TiO2 NP, LPS exhibits enhanced and time-shifted pro-inflammatory effects. Thus, in assessing the hazard of NP in real life, the enhanced effects of adsorbed bioactive molecules should be taken into account

Shape-Related Toxicity of Titanium Dioxide Nanofibres.

Titanium dioxide (TiO2) nanofibres are a novel fibrous nanomaterial with increasing applications in a variety of fields. While the biological effects of TiO2 nanoparticles have been extensively studied, the toxicological characterization of TiO2 nanofibres is far from being complete. In this study, we evaluated the toxicity of commercially available anatase TiO2 nanofibres using TiO2 nanoparticles (NP) and crocidolite asbestos as non-fibrous or fibrous benchmark materials. The evaluated endpoints were cell viability, haemolysis, macrophage activation, trans-epithelial electrical resistance (an indicator of the epithelial barrier competence), ROS production and oxidative stress as well as the morphology of exposed cells. The results showed that TiO2 nanofibres caused a cell-specific, dose-dependent decrease of cell viability, with larger effects on alveolar epithelial cells than on macrophages. The observed effects were comparable to those of crocidolite, while TiO2 NP did not decrease cell viability. TiO2 nanofibres were also found endowed with a marked haemolytic activity, at levels significantly higher than those observed with TiO2 nanoparticles or crocidolite. Moreover, TiO2 nanofibres and crocidolite, but not TiO2 nanoparticles, caused a significant decrease of the trans-epithelial electrical resistance of airway cell monolayers. SEM images demonstrated that the interaction with nanofibres and crocidolite caused cell shape perturbation with the longest fibres incompletely or not phagocytosed. The expression of several pro-inflammatory markers, such as NO production and the induction of Nos2 and Ptgs2, was significantly increased by TiO2 nanofibres, as well as by TiO2 nanoparticles and crocidolite. This study indicates that TiO2 nanofibres had significant toxic effects and, for most endpoints with the exception of pro-inflammatory changes, are more bio-active than TiO2 nanoparticles, showing the relevance of shape in determining the toxicity of nanomaterials. Given that several toxic effects of TiO2 nanofibres appear comparable to those observed with crocidolite, the possibility that they exert length dependent toxicity in vivo seems worthy of further investigation