SESSION D - MONITORING AND TOXICITY OF NANOMATERIALS

We investigated whether the toxicity of nanosilver (AgENMs) is size-dependent, by comparing effects of particles with the same chemical composition, charge and coating but with different, sizes (50, 80, 200nm). Additionally, we studied the effect of charge and surface composition on toxicity, using AgENMs of the same size, shape and specific surface area, but with different charges and surface compositions. AgENM uptake and subcellular localisation, dissolution and aggregation in protein-rich media, were also investigated.
Uptake and localisation, cell proliferation, cytotoxic effects, DNA damage (strand breaks and oxidised DNA lesions were assessed in A549 and TK6 cells, and the mutagenic potential of AgENMs in V79-4 cells. Physico-chemical characterization of the ENMs was performed in water/solvents and in biological media as a prerequisite to assessment of their impacts on cells.
All AgENMs were cytotoxic and genotoxic. However, 200nm AgENM was the most mutagenic. Regarding surface properties, positively charged AgENMs had greater impact on cytotoxicity/genotoxicity than did Ag ENMs with neutral or negative charge, likely to be related to their presence in the nucleus, implying a direct interaction with DNA.
Summarising, we demonstrated that AgENMs are potentially genotoxic and mutagenic and can induce different DNA lesions directly by contact with chromatin or DNA, or indirectly by reactive oxygen species, as indicated by DNA oxidation.
We suggest that expression of concentrations of ENMs as number of ENMs or surface area of ENMs (per cm2) is the most meaningful way to compare the toxicity of nanomaterials.
ACKNOWLEDGEMENTS:
Supported by the ECFP7 NanoTOES (PITN-GA-2010-264506), and NANoREG, (NMP.2012.1.3-3-310584).

Biological identity of a nanomaterial after the initial contact with a biological system strongly depend on the interaction between surface of a nanomaterial and a biological system like plasma or plasma membranes. Depending on the affinity of various proteins and lipids, a nanoparticle can coat with protein and/or lipids and exchange those at any later time as well. Here, fluorescence microspectroscopy (FMS), FRET concept and EDS TEM are employed to identify molecular contact between the lipids and surface of a nanomaterial where the affinity of lipids to nanomaterial surface is responsible for lipid wrapping around nanomaterial leading to a destabilization of a lipid bilayer.

The work focuses on qualitative and quantitative assessment of the DNA damage and cytotoxicity induced by CeO2 and TiO2 nanoparticles in the absence of photoactivation. The samples differ in the values of specific surface area and particle sizes (CeO2: 50 nm, 25 nm; TiO2: 22 nm, 19 nm) or, in case of TiO2, also in the crystal form (anatase/rutile, anatase). Human lung adenocarcinomic cell line (A549) was used as an experimental model. The cells were exposed to concentrations of 1000, 500, 100, 10 and 1 μg/ml of nanoparticles for 3 and 24 hours. The level of DNA damage was determined by the Comet assay. The percentage of DNA in a tail of the comet was applied as the parameter for the DNA damage quantification. Cytotoxicity was assessed by the trypan blue viability assay and the MTT cell proliferation assay. The results suggested that the CeO2 and TiO2 nanoparticles induced the DNA damage in A549 cells. The degree of DNA damage after an 3-hour exposure was higher than after a 24-hour period. Viability of A549 cells treated by nanoparticles for 24 hours significantly decreased. Biological effects of the nanoparticles depended on the particle size, dose and exposure time.

Acknowledgement
This project was supported by the following projects: SGS OU grant no. 31700/6159/1610/021185, Institute of Environmental Technologies (CZ1.05/2.1.00/03.0100), OP VaVpI, and NUP I (LO1208) and MH CZ-DRO–FNOs/2015.

Biokinetics of manganese oxide (MnO.Mn2O3) nanoparticles (NPs) was studied in male ICR mice after 17-week exposure in inhalation chamber by transmission electron microscopy (TEM), scan electron microscopy (SEM) and electrothermal atomic absorption spectrometry (ET-AAS) analyses. The study focused on the distribution of Mn NPs in selected tissues (lung, erythrocytes, liver, spleen, brain). First, uniformity, shape and size of NPs, used for exposure inhalation experiments, were characterized by TEM. The NP size was on average 20 nm (9-35 nm), and they mostly polygonal shape. Using ET-AAS analysis, we evaluated concentrations of Mn within individual organs; with exception of the lungs at the beginning of the exposure, and a slight increase of Mn levels in brain, no significant accumulation of Mn was observed. Next, the ultrathin sections of different tissues (which were not contrasted) were prepared and then observed using TEM and SEM. Mn NPs were found in lung alveoli, lung capillaries and pneumocytes, as well as in erythrocytes and alveolar macrophages; in addition, Mn NPs were detected in hepatocytes, Kupffer cells, spleen reticulocytes, medulla oblongata and in frontal cortex neuronal cells. The results indicate that Mn NPs may pass into alveoli and then through the wall of lung capillaries, through pneumocytes and endothelila cells into erythrocytes, which may distribute them into distant organs. This is supported by observation that in blood, a major portion of Mn NPs was found in erythrocytes, but not in white blood cells or plasma. Deposited Mn NPs were found mostly in secondary lysosomes of target cells. Together, our findings support the hypothesis that Mn NPs deposition after inhalation exposure occurs via lung-blood-target organ axis and that erythrocytes are major carriers of NPs.
ACKNOWLEDGEMENTS:
This study was supported by the Czech Science Foundation, project no. P503-12-G147.

We investigated toxic effects of extractable organic matter (EOM) from size fractions (1 – 10 µm; upper accumulation, 0.5 – 1 µm; 0.17 – 0.5 µm; ultrafine, < 0.17 µm) of particulate matter (PM) collected in Ostrava, Czech Republic, in acellular calf thymus (CT) DNA system and in A549 cells. For the CT-DNA system we obtained 26 samples corresponding to individual sampling days; for the A549 model we pooled the samples into groups representing inversions, periods between inversions and warm weather. EOMs were incubated 24h with CT DNA and A549. DNA adducts, oxidative damage and the aryl hydrocarbon receptor (AhR)-mediated activity were analyzed.
In the CT-DNA system, the upper accumulation fraction contained 44% of total carcinogenic polycyclic aromatic hydrocarbons (c-PAHs); the ultrafine fraction contained 11%. The highest DNA adduct levels were observed after incubation with the upper accumulation fraction. The AhR activity was driven by c-PAHs. Oxidative DNA damage was not related to c-PAHs. In the A549 model, the cumulative inversion sample normalized per volume of air contained the highest total PM and exhibited higher genotoxicity compared to other samples. When normalized to PM mass, ultrafine fraction was the most toxic in all measured parameters.
In conclusion, the upper accumulation fraction exhibited the most pronounced genotoxic effects. However, normalization to PM mass emphasized the significant toxic potential of ultrafine fraction. Thus, despite the low contribution of ultrafine fraction to a total PM mass in aerosol, it represents a serious health risk. Supported by Ministry of Education (LO1508) and the Czech Science Foundation (P503-12-G147).

Experimental in vitro and in vivo testing in modern toxicology might be significantly supported by the application of computational modeling (in silico studies). Such strategy helps to reduce cost, time and the use of laboratory animals.1, 2
Among various in silico techniques there are: (i) methods based on classical mechanics (i.e. molecular mechanics and molecular dynamics); (ii) methods based on quantum mechanics (i.e. ab initio, semiempirical and Density Fucntional Theory calculations); and (iii) chemoinformatic methods (i.e. Quantitative Structure-Activity Relationships modeling, grouping and read-across).1, 3, 4
Because of specific features and behavior of nanoparticles, computational methods originally developed for regular chemicals cannot always be applied explicite in nanotoxicology. Necessary adjustments have to be made.2, 5, 6 The lecture will discuss the current state-of-the-art and future needs in the development of computational modeling techniques for nanotoxicology.
REFERENCES:
1. Puzyn, T.; Leszczynska, D.; Leszczynski, J., Toward the Development of "Nano-QSARs": Advances and Challenges. Small 2009, 5, (22), 2494-2509.
2. Gajewicz, A.; Rasulev, B.; Dinadayalane, T. C.; Urbaszek, P.; Puzyn, T.; Leszczynska, D.; Leszczynski, J., Advancing risk assessment of engineered nanomaterials: Application of computational approaches. Adv Drug Deliver Rev 2012, 64, (15), 1663-1693.
3. Puzyn, T.; Rasulev, B.; Gajewicz, A.; Hu, X. K.; Dasari, T. P.; Michalkova, A.; Hwang, H. M.; Toropov, A.; Leszczynska, D.; Leszczynski, J., Using nano-QSAR to predict the cytotoxicity of metal oxide nanoparticles. Nat Nanotechnol 2011, 6, (3), 175-178.
4. Gajewicz, A.; Cronin, M. T.; Rasulev, B.; Leszczynski, J.; Puzyn, T., Novel approach for efficient predictions properties of large pool of nanomaterials based on limited set of species: nano-read-across. Nanotechnology 2015, 26, (1), 015701.
5. Tantra, R.; Oksel, C.; Puzyn, T.; Wang, J.; Robinson, K. N.; Wang, X. Z.; Ma, C. Y.; Wilkins, T., Nano(Q)SAR: Challenges, pitfalls and perspectives. Nanotoxicology 2014, 1-7.
6. Mikolajczyk, A.; Gajewicz, A.; Rasulev, B.; Schaeublin, N.; Maurer-Gardner, E.; Hussain, S.; Leszczynski, J.; Puzyn, T., Zeta Potential for Metal Oxide Nanoparticles: A Predictive Model Developed by a Nano-Quantitative Structure-Property Relationship Approach. Chem Mater 2015, 27, (7), 2400-2407.

Uniqueness and diversity of NMs raise questions about their safety, as well as about feasible ways to assess their potential toxicity. Despite intensive nanotoxicological research, it is impossible to keep pace with the rate of development and production of new NMs. Modifications of NMs size, shape and numerous other characteristics are performed to reach desirable industrial properties. To be able to alter, or rather minimize, toxicity of NMs in the same way, we have to understand which NMs properties govern particular toxic effects. Computational modeling relating physico-chemical properties of NMs with their toxic effects holds great promise in this area, however the lack of high-quality comparable experimental data of properly characterized NMs hampers the development of reliable models.
In the present study, we evaluated cytotoxic potential of selected TiO2 nanomaterials differing in basic characteristics (crystal structure, size and shape) using three in vitro assays (MTS, WST-1, LDH) and two human lung cell lines A549 and BEAS-2B, representing suitable in vitro models for studying inhalation toxicity. TiO2 is one of the most frequently used nanomaterials. Although inert in the bulk form, some TiO2 nanoforms have been reported to elicit toxic response. Our study compares the results of cytotoxicity of several nanoTiO2 variants in relation to their physico-chemical properties. The main purpose of the study is generation of high-quality data for computational modelling of cytotoxicity (nano-QSAR).
ACKNOWLEDGEMENTS:
Supported by the Czech Ministry of Education, Youth and Sports (COST CZ, LD14002 and LO1508) and by internal grant of the Nanotechnology Centre, VŠB–TUO, (SP2015/172).

The modern trend in risk assessment of engineered nanoparticles includes predictive modeling to optimize the cost of toxicity experiments. The number of costly measurements is minimized by employing toxicity predictions, based on the structure and properties of similar materials which have already been assessed. Unlike modeling the structure-activity relationship of chemicals or bulk-form materials, modeling the toxic activity of nanomaterials is a challenging task. The nanomaterials have a more complex structure which needs to be described. Beyond the chemical structure of base compounds, there are higher supramolecular structures which the particles are organized in. Next, each nanoparticle has its own ontological structure of properties. This structure defines the equipment, design and preparation of a particle together with its empirical characterization such as size, surface and electrochemical potential. Henceforth, we propose to employ relational learning in the nanomaterial toxicity modeling. Unlike conventional machine learning algorithms, which have mainly been used for the (nano)structure-activity relationship modeling, the relational methods use the first-order logic to describe the particle. This is more natural data representation than widely-used attribute vectors, as the data consist of highly structured characterized particles. Moreover, the relational approach enables to define key properties of a particle which have potential toxic effect. Based on this rule, a new particle is prepared and assessed to refine the current model. This should lead to the optimal toxicity experiments and production of particles safe by design.
ACKNOWLEDGEMENTS:
The research is supported by the Czech Ministry of Education, Youth and Sports (Grants No. LD14002 and LO1508).

Nowadays, nanomaterials are highly integrated into our daily life. However, recent studies have shown obvious toxicity of some nanoparticles to living organisms, and their potentially negative influence on environmental ecosystems. The goal of the present study is to develop efficient QSPR models that allow predicting the ecotoxicological properties and effects of inorganic nanomaterials (metals and oxides) by using the Online Chemical Modeling Environment (OCHEM). Numerical data on toxicity of nanoparticles to different organisms have been taken from the literature and uploaded in the OCHEM database. The main characteristics of nanoparticles such as chemical composition of nanoparticles, average particle size, shape and information about the biological test species were used as obligatory condition for all properties in OCHEM. 15 QSPR models were compared by following the same procedure with different combinations of descriptors and machine learning methods. QSPR methodologies used Random Forests (WEKA-RF), k-Nearest Neighbors and Associative Neural Networks. The predictive ability of the models was tested through leave-one-out cross-validation, giving a q2=0.69-0.79 for regression models and total accuracies Ac=76-100% for classification models. Predictions for the external evaluation sets obtained accuracies in the range of 78-100% (for low/high toxicity classifications) and q2=0.70-0.79 for regressions. The method showed itself to be a potential tool for estimation of toxicity of new nanoparticles at early stages of nanomaterial development.
ACKNOWLEDGEMENTS:
NATO Science for Peace and Security Programme by means of grant EAP.SFPP 984401.

Cubosomes and hexosomes enveloping well-defined internal nanostructures are promising nanocarriers for entrapment of anticancer drugs intended for the intravenous delivery. Limited information is available on the influence of plasma components on nanostructural and morphological features of cubosome and hexosome dispersions, which may modulate their stability in the blood and their overall biological performance. Here, we have addressed the structural events occurring after prolonged incubation of an anticancer drug cisplatin-free and cisplatin-loaded dispersions based on phytantriol (PHYT) with human plasma. Through an integrated approach involving synchrotron small angle X-ray scattering (SAXS), cryo-TEM, and Nanoparticle Tracking Analysis (NTA), we have studied the time-dependent effect of human plasma (and the plasma complement system) on the integrity of the internal nanostructure, morphology and fluctuation in size distribution of these nanostructured aqueous dispersions. The results indicate that in the presence of plasma, the internal nanostructure of cisplatin-free nanoparticles undergoes transition from a biphasic feature (a bicontinuous cubic phase of the symmetry Pn3m co-existing with an inverted type hexagonal (H2) phase) to a neat hexagonal (H2) phase, which decreases the median particle size. These observations were independent of a direct effect by serum albumin and dispersion-mediated complement activation. The solubilization of cisplatin significantly affects the internal nanostructure of these nanoparticles. This study provides a basic understanding of the effect of biological milieu on nanoparticulate cubosomal and hexosomal formulations. However, thorough investigation is needed in the future in order to establish a link between the interaction of cubosomes and hexosomes with plasma components and their performance after administration.

The immense beneficial potential of manufactured nanomaterials (MNM) may be threatened by limits of MNM environmental, health and safety (EHS) aspects understanding. Despite the extensive research in the field of nanotoxicology and exposure to MNM, comprehensive knowledge for regulation is still missing. NANoREG is the first FP7 project looking for answers needed by authorities on EHS by linking them to a scientific evaluation of data and test methods. It aims to provide a tool box of relevant instruments for reasonable risk assessment and management of MNM.
There is already number of achievements behind NANoREG project. The questions and needs of regulatory relevance have been identified and grouped within three knowledge gaps: hazard related characteristics of MNM, standardized methods to determine them, nano-specific risk assessment approaches. Nevertheless, standard operation procedures for some MNM characterization, toxicity testing and exposure measurements were developed. Critical exposure scenarios for MNM within the key value chains were identified. Toxicity testing focused on long-term inhalation toxicity, genotoxicity and immunotoxicity have been carried out. The Czech Republic actively participates at in vitro screening methodology development to evaluate toxicity by inhalation, at harmonization of genotoxicity protocols, exposure measurements and modelling and development of decision tree for risk assessment.
Despite the progress achieved since now, there are still many challenges regarding safety aspects of NMs which have to be addressed in further research.