Carbon quantum dots (CQDs) are considered as potential alternative to classical metal – based semiconductor quantum dots (Qdots) because of advantages of colorful photoluminescence and prominent biocompatibility in particular (Yang et al, 2009, Zhao et al, 2015). Thanks to those features CQDs have been widely applied in bioimaging, sensing, optoelectronics, etc (Shen et Liu, 2016). Here, we worked with carbon dots with quaternary ammonium attached on the carbon core through the amide linker. This surface modification gives CQDs positive charge, which could dramatically influence cytotoxicity of cells. In this study we used series of cytotoxicity tests (live/dead viability/cytotoxicity, cell cycle measurement, reactive oxygen species assay, commet assay and fluorescent optical microscopy) in order to reveal an influence of positive charged CQDs on human mesenchymal stem cells (hMSC).
Cells were treated with various concentration of CQDs for 24 hours. Fluorescent microscopy imaging showed that even at concentration 50 µg/ml CQDs were visible as red fluorescent clusters inside hMSC. The key concentration in all measurements was 200 µg/ml, where it was observed a slight decrease in viability, increase in DNA damage and significant increase in ROS generation and cell cycle analysis showed a decrease in cell growth and proliferation.
These positive CQDs could be suitable fluorescent probe for stem cell labeling in cell therapy when using the concentration less than 200 µg/ml. REFERENCES: Shen, L. M., & Liu, J. (2016). New development in carbon quantum dots technical applications. Talanta, 156, 245-256, Yang, S. T., Wang, X., Wang, H., Lu, F., Luo, P. G., Cao, L., Meziani, M. J., Liu, J. H., Liu, Y., Chen, M., Huang, Y., & Sun, Y. P. (2009). Carbon dots as nontoxic and high-performance fluorescence imaging agents. The Journal of Physical Chemistry C, 113, 18110-18114, Zhao, A., Chen, Z., Zhao, C., Gao, N., Ren, J., & Qu, X. (2015). Recent advances in bioapplications of C-dots. Carbon, 85, 309-327
It has long been known that results of toxicity and ecotoxicity studies tend to be significantly affected by the behavior of nanomaterials in exposure media. Especially agglomeration and aggregation significantly affect the resulting toxicity. Therefore, our effort was focused on development of testing procedure for soil ecotoxicology applicable in common laboratory practice providing data which are not distorted due to hardly controllable processes occurring during the experiment. The agar gel was therefore employed as an exposure medium which should supposedly suppress the rate of agglomeration and ensure homogeneous distribution of tested nanoparticles in the whole medium volume. Among other advantage of using agar as exposure media belongs the possibility of secondary characterization of nanomaterials. In an attempt to mimic or approach the conditions to which nanomaterials are exposed after application into the soil, further components were added to the agar and distilled water, namely natural organic matter (NOM) and kaolin. To assess the impact of these components on the behavior of nanomaterials and their resulting ecotoxicity various concentrations of NOM and kaolin were tested. Finally, ecotoxicity of zinc oxide nanoparticle (ZnO-NPs) powder with the mean particle size diameter of 10 nm to the annelid Enchytraeus crypticus was tested in modified agar. The procedure started with the re-suspension of nanoparticles in distilled water with the addition of dispersant. Obtained colloid was subsequently mixed with hot agar gel containing varying concentrations of NOM and kaolin.
The development of microscopy and new technologies allowed mass increase in production of engineered nanomaterials (NMs), ie. materials with particle diameter between 1-100 nm. Engineered NMs have unique properties due to their shape, size and large surface area and their use in matters of daily necessities is still increasing. The most commonly used engineered NMs are metals and their oxides, carbon compounds and silicon. Although physical properties of these NMs are mostly well known, possible harmful effects are often not adequately investigated.
In this study genotoxicity of TiO2 [anatase form; particle size 5-6 nm; NM101 (Joint Research Centre)], SiO2 [synthetic amorphous silicon produced by precipitation; particle size 14-23 nm; NM200 (Fraunhofer)] and Ag [rods; particle size 100-200 nm width, 5-10 µm length; NM302 (Fraunhofer)] nanoparticles was tested on transformed human bronchial epithelial cells BEAS-2B. The analysis was performed using a widely used cytokinesis-block micronucleus assay which detects nonspecific DNA damage in cells visible as micronuclei. The cells were exposed to three concentrations of NMs (0.25, 2.5, 6.25 µg/cm2 for NM101 and NM302; 0.25, 0.625, 1.25 µg/cm2 for NM200) for 28 and 48 hours.
Although silver nanoparticles emit reactive Ag+ ions which directly affect the cells, genotoxic mechanism of TiO2 and SiO2 nanoparticles is unclear. Our results suggest that longer exposure time increases the genotoxicity of tested nanoparticles and that SiO2 (NM200) is the most genotoxic NM of tested NMs. TiO2 (NM101) showed no increase of micronuclei formation even after 48 h exposure.
Pollution associated to road traffic and its combustion processes are considerably discussed in recent years. Nowadays, attention is also paid to the non-combustion processes, such as braking. During braking nonairborne and airborne fraction is released to the environment. Manufacturers use materials with low environmental risks, but during braking new compounds can be created, due to the high temperatures and pressures. Both fractions as well contain considerable amounts of released nano-sized particles which may enter to the water environment and may pose risk to the living organisms. However, there are no standardized and unified procedures which could be used for the brake wear debris toxicity assessment.
Aim of the study was the evaluation of the acute aquatic toxicity of the airborne brake wear debris and the most commonly used binders (phenolic resins) in the friction composites for brake linings. Brake wear debris from low-metallic brake pads was collected after standard dynamometer test from pocket filter and characterized by Raman microspectroscopy and scanning electron microscopy. Phenolic resin as binder is material with the highest volume in the formulation of the friction composite and contains Hexamethylenetetramine (HMTA) which can be decomposed at the ammonia and formaldehyde in high temperatures. Three commercially available phenolic resins were tested in the initial state and as well after thermal treatment (160°C), which simulate process of manufacturing. Scanning electron microscopy and Fourier-transform infrared spectroscopy were used for characterization of phenolic resins. EC50 toxicity parameter for freshwater green algae detection organism Raphidocelis subcapitata was evaluated for all tested materials.
Organometal lead halide perovskite is relatively new material, which has proved to be a promising material for construction of solar cells for photovoltaic technology. However, little is known about the toxicity of perovskite nanomaterials. Therefore, we performed several experiments to evaluate potential ecotoxicity of perovskites on soil bacteria Pseudomonas putida. Three types of perovskites were tested (CH3NH3PbI3, CHNHNH3PbBr3, CH3NH3PbBr3), each of them containing toxic lead, which leaked from the material. Therefore we determined the concentration of leaked lead using inductively coupled plasma mass spectrometry (ICP-MS). The effect of perovskites and lead on bacterial metabolism and viability was evaluated using respirometry and fluorescence microscopy analysis. Three concentrations of perovskites were tested – 50, 100 and 500 mg/L and four concentrations of lead in form of Pb(NO3)2 (100, 200, 500 and 1000 mg/L) in diluted Soya nutrient broth medium. The bacterial respiration was negatively affected by 500 and 1000 mg/L of lead; however it was not affected when similar concentration that leaked from the perovskite (100 mg/L and 200 mg/L) was added. Moreover, none of the three perovskite materials caused significant toxic effect towards bacteria.
Nanomaterials are understood as chemicals in the REACH directive and approaches of “classical” toxicology generally applied. Nevertheless, some properties of nanomaterials differ from compounds, e.g. size of basic unit, transport properties, corona etc. Growing use of nanomaterials will lead to their release to the environment and potential, even today unknown behaviour may appear. Human society has wide range of experience with unexpected or unknown toxic effects of engineered compounds (molecules), which may have parallel in nanoscale and which may demand development of new methods. Some examples are: Global pollution by lead in petrol anti-knock. Need of monitoring techniques for NMs in environment distinguishing ENM from natural NM is challenge arising from this parallel. Persistence, long-distance transport and bioaccumulation of POPs show us the necessity to understand and to measure transport properties, behaviour in living organisms an life cycle of ENMs. Mercury transformation to more toxic methylmercury (e.g. Minamata disease) is an example of environmental transformation with increased hazard and its parallel may be transformation of ENM to other, more toxic particles in environment, and even their ”invisibility”. Environmental fate studies will need both identification and monitoring technics, only rarely present today. New forms of toxicity and gaps in testing may be recognized in nanoscale, similarly as endocrine disruption or thalidomide effects were unexpected before their discovery. Since the questions about behaviour of nanomaterials in environment will be understood enough, already existing precautionary technics as life cycle analysis and cleaner production may be applied to minimize exposure to nanomaterials throw environment.
Engineered nanomaterials (ENMs) are applied in a wide range of products worldwide. The use of ENM results in a generation of increasing amounts of waste containing nanomaterials. Wastes of electronic and electrical equipment (e-waste) are one of the main waste streams recycled in municipal and industrial waste. There are limited studies that assess the potential exposure to nanomaterials during the recycling processes. The main way how is e-waste prepared for the following recycling is the reduction of its size.
The aim of the study was to carry out the preliminary risk assessment of nanomaterials generating within e-waste processing. Occupational exposure was measured in a worker's breathing zone at the recycling site by Condensation Particle Counter (CPC 3007). Workplace air samples were collected on glass microscope slides with Nano-ID Select Wide Range Aerosol Sampler (NANO-ID Select 005) and analysed by Scanning Electron Microscopy (SEM) and Energy Dispersive X-Ray Spectroscopy (EDS). The qualitative risk assessment has been carried out in CB Tool v 2.0. The highest concentrations of nanoparticles were detected for the following elements: chromium, copper, zinc, and tin. The mean concentration of nano- and ultrafine particles (range of 10 - 1000 nm) was 65792 particles/cm3 for all particles. The level of risk obtained from CB tool was equal to RL2, and RL3. Local exhaust ventilation was proposed, based on the assessed risk level, to ensure occupational health and safety.
This field study has been conducted as a case study for the subsequent detailed risk assessment and management.
Challenging field of live-imaging by nanoscale particles has undergone profound research in recent years. Semiconductor quantum dots (QD) have emerged as possible biomedical imaging tool because of their natural bright photoluminescence (PL), water solubility and particularly for their diameter size approximating the size of biomolecules (DNA, proteins). However, even though heterogenous semiconductor QDs possess remarkable physico-chemical properties, their usage for biological purposes is limited to the in vitro research due to serious concerns about their potential toxicity for living organisms. In contrast, QD consisted of silicon (SiQD) attract the attention as a low-toxicity material suitable for imaging studies even on the in vivo level.
In this study, SiQDs co-doped with boron and phosphorus were used for in vitro evaluation of their complex influence on human osteoblasts. SiQDs (4 nm, peak emission 800 nm) display long lasting dispersion in methanol and aqueous solutions and PL insensitive to the changes of environment and surface modifications. Cells were exposed to the increasing concentrations (25 – 125 µg/ml) of SiQDs in the presence or absence of proteins from fetal bovine serum (component of cultivation medium) and their cytotoxicity was evaluated after 6, 24 and 48 hours. The ways of internalization of SiQDs into the cells were observed and evaluated via confocal laser scanning microscope. The time-lapse experiments of SiQD uptake were performed with the relation to the presence/absence of proteins. Description of SiQD optical parameters and evaluation of zeta potential led to more complex understanding of observed in vitro findings.
General population is exposed to exhaust fumes resulting from the combustion of diesel fuels. Their toxicity is influenced by a number of factors, including diesel exhaust particles (DEPs) sizes. Until recently, it was claimed that the toxic effect of DEPs is related to particles with a diameter less than 2.5 µm. However, newest reports suggest that the most harmful effects are caused particles in sizes not exceeding 0.2 µm. DEPs causes harmful changes in the respiratory and cardiovascular systems, they are also known mutagen and carcinogen substances (1A IARC group). Although the mechanism of DEPs carcinogenic action is not fully understood, it is known that DEPs can penetrate biological membranes, inducing an inflammatory response and interfering with the cells oxygen metabolism. It was also postulated that DEPs can lead to the induction of EMT and metastasis, a major cause of cancer deaths. One of the mechanisms of metastasis is epithelial-mesenchymal transition (EMT), during which comes to reorganization of the cells actin cytoskeleton. Taking into account these reports, the aim of this project was to investigate the biological activity of nanoparticles obtained after combustion of B7 diesel fuel on a breast cancer derived MDA-MB-436 cells. MDA-MB-436 cells are well-characterized model for study of metastasis of malignant tumors. The toxicity of the particles were examined by two colorimetric assays - using neutral red dye and a tetrazolium salt reduction. In further experiments concentrations not exceeding the IC50 value were used. Subsequently, assessment of changes in the organization of the actin skeleton of cells treated with DEPs was determined by staining with fluorochrome-labeled phalloidin. Conducted research seem to be significant in view of the high cancer mortality and diesel engines popularity.
ACKNOWLEDGEMENTS: This study was funded by the Ministry of Science and Higher Education basis on the decision number MN 16001 and from Norway Grants in the Polish-Norwegian Research Programme operated by the National Centre for Research and Development: Pol-Nor/201040/72/2013 (FuelHealth).
The ubiquity of nanoparticles (NPs) in almost all areas of industry has resulted in their – mostly unintentional – widespread release into the environment. The potential hazard is closely related with tier size, because as with the decreasing size increases the ability to cross the biological barriers and penetrate into living organisms, and consequently, causing a toxic effect.
Presently, the precise risk assessment of nanoparticles in the environment is impossible because of the lack effective, analytical methods, which could characterize NP mobility, persistence and reactivity and in consequence reall ecotoxicity. In order to avoid the hazards of nanoparticles by significantly reducing exposure, it is necessary to developed effective nanoparticles fate monitoring system. So, it is strictly necessary to know every mechanism determining the tendency of nanoparticles for aggregation/agglomeration and/or their disintegration by identifying factors influencing the stability of suspension of nanoparticles. Tool for predicting physicochemical properties of nanoparticles, such as zeta potential or dimension aggregated/agglomerated structures in different solutions, will allow estimating potential exposure, especially forming the single and smallest nanoparticles posing the highest risk.
The main objective of the project is to extend the knowledge and defining the crucial factors impacting fate of the nanoparticles in the natural environment, including the dynamic self-assembly process into larger structures, by combination of experimental and computational methods using QSPR approach. The project verifies the hypothesis according to which, ions affect the dynamic self-assembly of NPs into larger aggregated/agglomerated structures or their disaggregation in completely different way due to the differential physicochemical characteristics.
In production of cosmetics and pharmaceuticals, a product safety assessment is one of the crucial steps in the process before distribution to the market.
Presented work is focused on characterization of chosen natural extracts in terms of the fenolic compounds content and antioxidant activity. These extracts were encapsulated into liposomes and chitosan nanoparticles. The aim was testing possible genotoxicity of the extracts and particles using model organism – bacteria E. coli.
Total phenolics, flavonoids and antioxidant activity was evaluated by spectrophotometrical methods as well as encapsulation efficiency. Size of prepared particles was determined by dynamic light scattering and stability of particles was determined using zeta potential. To analyse genotoxic effect SOS Chromotest was used.
In this work it was found that neither pure extracts nor nanoparticles show genotoxic effect on model prokaryotic organism. Nanoparticles with encapsulated active compounds can enhance the effectiveness of cosmetics and pharmaceuticals. Nevertheless, further investigation is neccessary.
Nowadays, nanomaterials are widely used in industry, medicine, cosmetics, remediation of persistent pollutants, and therefore it is important to test their possible harmful effects on both human health and natural environment. However, nanoparticles can be often contaminated by bacterial endotoxins (lipopolysaccharides, pyrogens) that can cause false positive results of the (eco)toxicological tests. For this reason, nanoparticle samples should be always screened for endotoxin presence before performing any of these tests. Many types of nanoparticles interfere with traditional methods determining the level of endotoxin contamination. The aim of our study was to evaluate two different methods for endotoxin detection and to compare their performance. The first commonly used method, chromogenic LAL assay, is fast, and relatively cheap, but it is known for the interference of the nanoparticles with the test. The second one, EndoLISA, is a new, ELISA-based assay causing no interference of nanoparticles with the assay due to specific binding of endotoxin onto a microtiter. Our results demonstrate that EndoLISA assay can be less sensitive than chromogenic LAL assay in low endotoxin concentrations, but it is more accurate in higher endotoxin levels.
Nowadays, road traffic and related traffic pollution are in forefront of the interest. Settled road dust is a complex material which contains particles from natural and anthropogenic sources. Particles present in the road dust could be nano-sized, thus could be easily transferred to the water environment and/or resuspended to the air. Nano-sized particles due to the small sizes and weights can be spread for the long distances by air flows and may stay long time suspended in the air. So these particles can be easily inhaled or ingested. Human saliva and nose sputum are the first barriers of the respiratory tract in the human organism. Our previous study confirmed presence of metal based particles which could be connected with the traffic pollution in the human nasal mucosa.
The aim of the study was to determine the presence of metal based particles in the human saliva from two donors after an hour walk nearby busy road in the city of Ostrava. For the comparison saliva was collected before and after one hour walk nearby busy road. As well road dust from the same busy road was collected and prepared as leachate in artificial saliva solution to compare settled and suspended road dust in human saliva. Collected road dust and as well the obtained samples of saliva were characterized by scanning electron microscopy to determine metal based particles. In the sample of artificial saliva and human saliva after walk were detected metal based particles which do not have biogenic origin.
Brake processes have been extensively studied and discussed in the relation to the creation of the brake wear. However, the contribution to the environmental pollution is still not clear and there is not unified procedure for the evaluation of the potential risk of the brake wear to the environment and human health. Lipid peroxidation, which could be defined as the oxidative deterioration of lipids containing any number of carbon-carbon double bonds especially polyunsaturated fatty acids, is one of the markers of oxidative stress and linoleic acid as a model lipid of cell membrane is used to quantifying the potency of tested compounds to induce peroxidation damage (lipoperoxidation) of unsaturated lipids.
The aim of the study is evaluation of the potential toxicity of brake wear debris and typical constituents used in formulation of friction composites for brake linings by cell-free chemical test. For quantifying of lipoperoxidation was used test based on the reaction of malondialdehyde (natural product of lipid peroxidation) with thiobarbituric acid to produces pink adduct. Brake wear debris from commercial available brake pads and reference friction composites were collected after standard brake dynamometer test. Lipoperoxidation was evaluated for the collected brake wear debris and for chosen compounds commonly used in brake formulations (titanate, barite, iron fibers, bronze fibers, and chromite). Both types of brake wear debris were analyzed by scanning electron microscopy and Raman spectroscopy. Experimental results showed slightly toxic character for chromite, iron fibers, barite, and brake wear from reference friction composite.
Nanostructures in general, include quantum dots (QDs), have great potential for use in many applications, technical, biological and medical. Despite their great potential, there is an enormous disadvantage for quantum dots containing some metalloid elements and heavy metals especially. This disadvantage is caused by the gradual release of metal ions and instability of QDs in different environments. Covering QDs by various modifiers is usually used for QDs stabilization and decrease of heavy metals release. The accomplishment of desired effect and efficiency of QDs modifications is questionable. In the present study, ODs stability via their fluorescent properties, size and release of cadmium ions were studied. To measure the release of Cd2+ from the core of QDs an electrochemical method, differential pulse voltammetry, was used. Study conducted in mildly alkaline pH, which was close to the actual pH of QDs was performed. Four types of QDs were used: CdTe / ZnSe (core/shell), MPA - CdTe, MSA - CdTe, GSH - CdTe. During 25 days period measurement of fluorescence, particle size and release of Cd2+ ions from QDs in two alkaline buffers were performed. Fist was phosphate buffer pH 7.6 and and further used a special neutral buffer pH 7.6, which simulated the cellular environment. From these data the stability of given modified and core shell QDs were assessed. At the end of the study it was found that ZnSe / CdTe core shell QDs maintain high fluorescence values together with the lowest released cadmium ions concentration.
The growing industrial, health care, pharmaceutical and water purification application of nanoparticles (NP’s) has upraised concerns about their effect on the human health and environment. Whether the NP’s dissolve with the release of ions, or coarsen to form larger aggregates, is crucial in finding out their possible fate. Herein, the stability of tannic acid/sodium citrate-stabilized Ag and Silicon-Graphite NP’s in water was investigated at various pH values and ionic strengths (IS). In order to test it, three measurement methods were applied, namely disc centrifuge analysis, UV-Vis spectrophotometry and zeta potential analysis. The kinetics of ion release and/or NP agglomeration was strongly pH- and IS- dependent which resulted in change of UV-Vis spectra, particle size distribution and particle’s zeta potential. Low pH resulted in a fast dissolution of Ag NP’s whereas a high pH did not affect them significantly. In contrary, high pH has strongly influenced the stability of Si-Graphite NP’s. Moreover, the stability of NP’s was assessed in the water sample collected from Harcov reservoir. It was found that the organic matter and ions present therein significantly influenced both NP’s causing their rapid agglomeration and/or dissolution. Furthermore, the effect of these NP’s in deionised and Harcov reservoir water on the soil bacterium Pseudomonas putida was assessed.
Dynamic light scattering (DLS) is a technique for nanoparticle size characterization in a liquid environment. The integrated EU project NANoREG – “A common European approach to the regulatory testing of Manufactured Nanomaterials” - provides diverse well characterized nanoparticles in order to compare toxicity results among EU laboratories. Two state-of-art DLS instruments were available in our laboratory – a NanoBrook Omni (Brookhaven Instruments Corporation, U.S.A.) and a Zetasizer Nano (Malvern Instruments Ltd., U.K.). Comparable tests were performed on standard nanoparticles (NANOSPHERE™ Size Standards, Thermo Scientific) and batch samples (2.56mg/ml) of the NANoREG project nanoparticles under identical experimental conditions. The standard was measured undiluted and gradually diluted by ultrapure water. NANoREG particles were diluted according to the standard operating procedure developed within NANoREG . The standard and NANoREG particles were measured by back-scattering to prevent issues arising from sample opacity. Standard size was correctly measured in undiluted form by the Zetasizer Nano; the NanoBrook Omni was able to measure the right size at 0.4mg/ml concentration and lower. This is uncomfortable for the operator despite the fact that the dilution itself certainly affects the extent of aggregation of the suspended nanoparticles. NANoREG samples revealed that differences among measured sizes by the instruments increase with rising sample opacity. We concluded that under identical experimental conditions, the Zetasizer Nano is suitable for quick and reliable DLS measurements of nanoparticle suspensions, while the NanoBrook Omni requires dilution of more concentrated suspensions, which makes data useless for undiluted samples tested for toxicity. Support: Research Infrastructure NanoEnviCz, Project No. LM2015073.