BackgroundMassive industrial production of engineered nanoparticles poses questions about health risks to living beings. In order to understand the underlying mechanisms, we studied the effects of TiO2 and ZnO agglomerated engineered nanoparticles (EPs) on erythrocytes, platelet-rich plasma and on suspensions of giant unilamelar phospholipid vesicles.ResultsWashed erythrocytes, platelet-rich plasma and suspensions of giant unilamelar phospholipid vesicles were incubated with samples of EPs. These samples were observed by different microscopic techniques. We found that TiO2 and ZnO EPs adhered to the membrane of washed human and canine erythrocytes. TiO2 and ZnO EPs induced coalescence of human erythrocytes. Addition of TiO2 and ZnO EPs to platelet-rich plasma caused activation of human platelets after 24 hours and 3 hours, respectively, while in canine erythrocytes, activation of platelets due to ZnO EPs occurred already after 1 hour. To assess the effect of EPs on a representative sample of giant unilamelar phospholipid vesicles, analysis of the recorded populations was improved by applying the principles of statistical physics. TiO2 EPs did not induce any notable effect on giant unilamelar phospholipid vesicles within 50 minutes of incubation, while ZnO EPs induced a decrease in the number of giant unilamelar phospholipid vesicles that was statistically significant (p < 0,001) already after 20 minutes of incubation.ConclusionsThese results indicate that TiO2 and ZnO EPs cause erythrocyte aggregation and could be potentially prothrombogenic, while ZnO could also cause membrane rupture.

BackgroundWe studied the effect of carbon black (CB) agglomerated nanomaterial on biological membranes as revealed by shapes of human erythrocytes, platelets and giant phospholipid vesicles. Diluted human blood was incubated with CB nanomaterial and observed by different microscopic techniques. Giant unilamellar phospholipid vesicles (GUVs) created by electroformation were incubated with CB nanomaterial and observed by optical microscopy. Populations of erythrocytes and GUVs were analyzed: the effect of CB nanomaterial was assessed by the average number and distribution of erythrocyte shape types (discocytes, echinocytes, stomatocytes) and of vesicles in test suspensions, with respect to control suspensions. Ensembles of representative images were created and analyzed using computer aided image processing and statistical methods. In a population study, blood of 14 healthy human donors was incubated with CB nanomaterial. Blood cell parameters (concentration of different cell types, their volumes and distributions) were assessed.ResultsWe found that CB nanomaterial formed micrometer-sized agglomerates in citrated and phosphate buffered saline, in diluted blood and in blood plasma. These agglomerates interacted with erythrocyte membranes but did not affect erythrocyte shape locally or globally. CB nanomaterial agglomerates were found to mediate attractive interaction between blood cells and to present seeds for formation of agglomerate - blood cells complexes. Distortion of disc shape of resting platelets due to incubation with CB nanomaterial was not observed. CB nanomaterial induced bursting of GUVs while the shape of the remaining vesicles was on the average more elongated than in control suspension, indicating indirect osmotic effects of CB nanomaterial.ConclusionsCB nanomaterial interacts with membranes of blood cells but does not have a direct effect on local or global membrane shape in physiological in vitro conditions. Blood cells and GUVs are convenient and ethically acceptable methods for the study of effects of various substances on biological membranes and therefrom derived effects on organisms.

Extracellular vesicles (EVs) are membrane-enclosed fragments shed from all cell types, including tumour cells. EVs contain a wide range of proteins, biolipids and genetic material derived from mother cells and therefore may be potential biomarkers for tumour diagnosis, disease progression and treatment success. We studied the effect of canine mast cell tumours (MCTs) on EV concentrations in blood isolates in association with MCT's histological grade, Ki-67 proliferative index, KITstaining pattern and number of PLT. The average EV concentration in blood isolates from nine dogs with MCTs was considerably higher than that in blood from eight healthy dogs. But there were no statistically significant differences in EVs concentration in the population of dogs with MCT according to a different histological grade of malignancy (Patnaik, Kiupel), KIT-staining pattern and Ki-67 proliferation index. The results show that these variables statistically do not significantly predicted EV concentrations in blood isolates (P > .05), except the KIT-staining pattern I which added statistically significantly to the prediction (P < .05). The results confirmed the impact of neoplasms on the morphological changes to cell membranes, which result in greater vesiculability and higher EV concentrations. K E Y W O R D S canine, extracellular vesicles, mast cell tumour, oncology, small animal

BackgroundRecent advances in development and industrial production of nanomaterials require assessment of their effects on human and animal health. Toxicity of nanomaterials has therefore become an issue regarding the question which methods are the most appropriate for determining health risk [1,2]. Nanomaterial introduces also effects that cannot be explained by composition of the material and chemical reactions, but require consideration of non-specific properties, such as size and shape of particles, their electromagnetic properties and interactions with biological material [3]. Since the underlying mechanisms are largely unknown, standard methods for research, testing and safety are not necessarily relevant for these materials. Standard methods for research and testing of various compounds include experiments on experimental animals. These methods were found to have poor predictability regarding other species, in particular human, as shown by carcinogeneity studies [4][5][6]. It was suggested that in vitro research may provide essential information pertaining to the human health risks posed by nanoparticle exposure [7]. With fast and extensive development of new nanomaterials and their use in medicine and industry there is urgent need to develop effective and low cost methods that will enable understanding basic processes in living organisms. The role of nonspecific biophysical mechanisms has hitherto been underestimated as potentially essential in revealing biological processes but should be considered in future paradigms of research and testing of nanomaterials.Exogenously added substances first come in contact with cells by interacting with the membrane, so it is of great interest to study the interaction of these substances with biological membranes. Convenient systems for such studies are mammalian erythrocytes and giant unilamelar phospholipid vesicles (GUVs) composed of a closed bilayer membrane. These entities do not have internal structure (other than cortical membrane skeleton in case of erythrocytes) so their equilibrium shape is determined by the minimum of the membrane free energy [8]. Interaction of the added substance with the membrane causes changes in the membrane properties and in the constraints imposed upon the cell/vesicle (fixed area of the membrane, fixed difference between the areas of the two membrane layers, fixed cell/vesicle volume) which is reflected in the shape change [9]. Since mammalian erythrocytes and phospholipid vesicles (sized up to 100 micrometers) can be observed under the optical microscope, the effect of the exogenously added substances can be directly followed in real time.Further, of special interest are processes caused by exogenously added substances that increase the risk for thromboembolic events [ 10]. Some parameters of these processes, e.g. activation of platelets and coalescence of membranous structures due to the presence of nanomaterial have previously been studied [11].Membrane -nanomaterial interactions could be considered as one of the basic elements i...

Clinical studies have indicated that the NV (nanovesicle) concentration in blood samples is a potential indicator of clinical status and can be used to follow the development of the disease. For 32 months, we monitored the effect of imatinib treatment on NV concentrations in blood samples from 12 patients with GIST (gastrointestinal stromal tumour). The NV concentration before the treatment increased with respect to control by a factor of 3.5 on average (range 2.6-9.2). The first week after initiation of the treatment, the NV concentration increased considerably, by a factor of 13 on average (range 5.9-21.2), whereas on average, after 1 month, it decreased to the level of the control and remained at that level for at least 1.5 years. Recent assessment (after 2.5 years) showed a somewhat increased NV concentration, by a factor of 2 on average (range 0.7-3.9). Low NV concentrations in blood samples during the treatment reflect a favourable effect of imatinib in these patients and no remission of the disease was hitherto observed.