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Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production.

The establishment of reliable and robust in vitro models for hazard assessment, moving away from animal testing, requires evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as monoculture and, to increase the relevance of the model, also as co-culture with macrophages (derived from the THP-1 monocyte cell line or from human blood monocytes). Exposure employed the VITROCELL® Cloud12 system at physiologically relevant doses. Overall, the results of the seven participating laboratories are quite similar. No effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 exposure on cell viability and barrier integrity were seen in the monoculture and co-culture models. Most laboratories showed that LPS induced cytokine release in the co-culture models, but not the monoculture model. Exposure to DQ12 or TiO2 did not increase cytokine release, probably due to too low deposited doses. Interlaboratory variability indicated acceptable variation for cell viability measurements and relatively high variation for cytokine measurements. Based on the transferability and reproducibility of the model and its air exposure we are confident that, based on detailed SOPs, performing a new round of interlaboratory comparison, and increasing the deposited doses of NMs, these models may be taken further towards a possible OECD guideline.

Nanoparticles (NPs) have attracted considerable attention in various fields, such as cosmetics, the food industry, material design, and nanomedicine. In particular, the fast-moving field of nanomedicine takes advantage of features of NPs for the detection and treatment of different types of cancer, fibrosis, inflammation, arthritis as well as neurodegenerative and gastrointestinal diseases. To this end, a detailed understanding of the NP uptake mechanisms by cells and intracellular localization is essential for safe and efficient therapeutic applications. In the first part of this review, we describe the several endocytic pathways involved in the internalization of NPs and we discuss the impact of the physicochemical properties of NPs on this process. In addition, the potential challenges of using various inhibitors, endocytic markers and genetic approaches to study endocytosis are addressed along with the principal (semi) quantification methods of NP uptake. The second part focuses on synthetic and bio-inspired substances, which can stimulate or decrease the cellular uptake of NPs. This approach could be interesting in nanomedicine where a high accumulation of drugs in the target cells is desirable and clearance by immune cells is to be avoided. This review contributes to an improved understanding of NP endocytic pathways and reveals potential substances, which can be used in nanomedicine to improve NP delivery., This review contributes to the current understanding of NPs cellular uptake and gives an overview about molecules, which can enhance or decrease cellular internalization of NPs.

Understanding the molecular responses of plant roots to the challenging environment contributes to engineering plants with improved stress tolerance. However, little has been done to understand rice (Oryza sativa L.) roots response to the toxic concentration of zinc (Zn) at the proteomic level. This study explored proteomic responses of young rice roots 5–6 days after sowing to 765 μM of Zn in a hydroponic set-up after 4-day exposure. Dye staining method and spectrophotometry were chosen for physiological response investigations. ICP-MS was used to determine metal content in rice seedlings. Two-dimensional gel electrophoresis was used for the proteomic study of root tissue. Elevated Zn reduced Mg translocation to the shoots and Mn uptake, decreased plant growth, and increased cell death of roots. Zn stimulated the biosynthesis of glutathione but decreased ROS and malondialdehyde levels. In total, 42 Zn-responsive proteins were identified. Proteins involved in redox regulation, defense response, sulfur metabolism, and proteolysis were induced, while those of energy production and cell wall biogenesis were decreased. Roots of O. sativa seedlings could tolerate certain Zn excess (765 μM Zn for 4 days) by stimulating nutrient recycling and glycolysis, and production of defensive metabolites and proteins to maintain ion and redox homeostasis. Besides, adenosylhomocysteinase (AHCY) down-regulation may contribute to balanced transmethylations, and methionine salvage pathway appears important for the adaptation to Zn. Our results provide some new insight into a complex metabolic response of rice roots to Zn stress, which is related to both stress and defense mechanisms.

Zala Jan,1 Mitja Drab,2 Damjana Drobne,3 Apolonija Bedina Zavec,4 Mojca Benčina,5 Barbara Drasler,3 Matej Hočevar,6 Judita Lea Krek,1 Ljubiša Pađen,1 Manca Pajnič,1 Neža Repar,3 Boštjan Šimunič,7 Roman Štukelj,1 Veronika Kralj-Iglič1 1Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia; 2Laboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, Slovenia; 3Nanobiology and Nanotoxicology Group, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia; 4Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Ljubljana, Slovenia; 5Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia; 6Physics and Chemistry of Materials, Laboratory of Surface Engineering and Applied Surface Science, The Institute of Metals and Technology, Ljubljana, Slovenia; 7Institute for Kinesiology Research, Science and Research Centre Koper, Koper, SloveniaCorrespondence: Veronika Kralj-IgličLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, Ljubljana, SloveniaEmail kraljiglic@gmail.comIntroduction: Cellular nanovesicles (CNVs), that are shed from cells, have been recognized as promising indicators of health status. We analyzed the effect of long-distance running on concentration of CNVs, along with some standard blood parameters, in 27 athletes two days before and > 15 hours after physical effort.Methods: CNVs were isolated by repetitive centrifugation and washing of samples, and assessed by flow cytometry. Cholinesterase (ChE) and glutathione S-transferase (GST) activity were measured spectrophotometrically. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) was measured with immunoturbidimetric determination and lipidogram parameters were measured by enzymatic colorimetric assay. Flow cytometry was used for blood cell count and mean platelet volume (MPV) measurement.Results: More than 15 hours after physical effort a decrease was found in CNVs’ concentration in isolates from blood (46%; p< 0.05), in ChE activity in whole blood (47%; p< 0.001), in plasma (34%; p< 0.01), and in erythrocyte suspension (54%; p< 0.001), as well as in GST activity in erythrocyte suspension (16%; p< 0.01) and in IL-6 concentration in plasma (63%; p< 0.05). We found no change in GST activity in plasma and in TNF-α concentration in plasma. Correlations (> 0.8; p< 0.001) between CNVs’ concentration and ChE activity, and GST activity, respectively, in erythrocyte suspension were found.Conclusion: We found that > 15 hours post-physical effort, CNVs’ concentration was below the initial value, concomitant with other measured parameters: ChE and GST activity as well as IL-6 concentration, indicating a favorable effect of physical effort on health status. CNVs’ concentration and ChE activity in isolates from peripheral blood proved to have potential as indicators of the response of the human body to inflammation after physical effort. Physical activity should be considered as an important factor in preparation of subjects for blood sampling in procedures focusing on CNV-containing diagnostic and therapeutic compounds.Keywords: membrane vesiculation, physical effort, blood samples, inflammation process, cellular nanovesicles, marathon

Tissue models mimic the complex 3D structure of human tissues, which allows the study of pathologies and the development of new therapeutic strategies. The introduction of perfusion overcomes the diffusion limitation and enables the formation of larger tissue constructs. Furthermore, it provides the possibility to investigate the effects of hematogenously administered medications. In this study, the applicability of hydrophilic polytetrafluoroethylene (PTFE) membranes as vessel-like constructs for further use in perfused tissue models is evaluated. The presented approach allows the formation of stable and leakproof tubes with a mean diameter of 654.7 µm and a wall thickness of 84.2 µm. A polydimethylsiloxane (PDMS) chip acts as a perfusion bioreactor and provides sterile conditions. As proof of concept, endothelial cells adhere to the tube's wall, express vascular endothelial cadherin (VE-cadherin) between neighboring cells, and resist perfusion at a shear rate of 0.036 N m-2 for 48 h. Furthermore, the endothelial cell layer delays significantly the diffusion of fluorescently labeled molecules into the surrounding collagen matrix and leads to a twofold reduced diffusion velocity. This approach represents a cost-effective alternative to introduce stable vessel-like constructs into tissue models, which allows adapting the surrounding matrix to the tissue properties in vivo.

Silica nanoparticles (NPs) are widely used in various industrial and biomedical applications. Little is known about the cellular uptake of co-exposed silica particles, as can be expected in our daily life. In addition, an inflamed microenvironment might affect a NP&rsquo, s uptake and a cell&rsquo, s physiological response. Herein, prestimulated mouse J774A.1 macrophages with bacterial lipopolysaccharide were post-exposed to micron- and nanosized silica particles, either alone or together, i.e., simultaneously or sequentially, for different time points. The results indicated a morphological change and increased expression of tumor necrosis factor alpha in lipopolysaccharide prestimulated cells, suggesting a M1-polarization phenotype. Confocal laser scanning microscopy revealed the intracellular accumulation and uptake of both particle types for all exposure conditions. A flow cytometry analysis showed an increased particle uptake in lipopolysaccharide prestimulated macrophages. However, no differences were observed in particle uptakes between single- and co-exposure conditions. We did not observe any colocalization between the two silica (SiO2) particles. However, there was a positive colocalization between lysosomes and nanosized silica but only a few colocalized events with micro-sized silica particles. This suggests differential intracellular localizations of silica particles in macrophages and a possible activation of distinct endocytic pathways. The results demonstrate that the cellular uptake of NPs is modulated in inflamed macrophages but not in the presence of micron-sized particles.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three- dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor α) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

BackgroundAir pollution is killing close to 5 million people a year, and harming billions more. Air pollution levels remain extremely high in many parts of the world, and air pollution-associated premature deaths have been reported for urbanized areas, particularly linked to the presence of airborne nano-sized and ultrafine particles.Main textTo date, most of the research studies did focus on the adverse effects of air pollution on the human cardiovascular and respiratory systems. Although the skin is in direct contact with air pollutants, their damaging effects on the skin are still under investigation. Epidemiological data suggested a correlation between exposure to air pollutants and aggravation of symptoms of chronic immunological skin diseases. In this study, a systematic literature review was conducted to understand the current knowledge on the effects of airborne particulate matter on human skin. It aims at providing a deeper understanding of the interactions between air pollutants and skin to further assess their potential risks for human health.ConclusionParticulate matter was shown to induce a skin barrier dysfunction and provoke the formation of reactive oxygen species through direct and indirect mechanisms, leading to oxidative stress and induced activation of the inflammatory cascade in human skin. Moreover, a positive correlation was reported between extrinsic aging and atopic eczema relative risk with increasing particulate matter exposure.

The understanding of how nanomaterials interact with cells, tissues, and organs is important to realize their potential for biomedical applications and for hazard assessment of occupational exposure. Depending on the nanomaterial characteristics, they can trigger various components of the immune system, that is, innate and adaptive immune systems, thereby either activating or suppressing immune system functions. In this chapter, we highlight the currently established immune cell models and readouts to address the possible effects of nanomaterials. In addition to the conventional 2D cultures, the design and use of more complex 3D models is emphasized.

The increased mass production of graphene related materials (GRM), intended for a broad spectrum of applications, demands a thorough assessment of their potential hazard to humans and the environment. Particularly, the paramount concern has been expressed in regard to their interaction with the respiratory system in occupational exposure settings. It has been shown that GRM are easily respirable and can interact with lung cells resulting in the induction of oxidative stress or pulmonary inflammation. However, a comprehensive assessment of potential biological effects induced by GRM is currently hardly feasible to accomplish due to the lack of well-defined GRM materials and realistic exposure data. Herein, a 3D human lung model was combined with a commercial aerosolization system to study potential side effects of GRM. Two representative types of GRM were aerosolized onto the lung epithelial tissue surface. After 24 h post exposure, selected biological endpoints were evaluated, such as cell viability, morphology, barrier integrity, induction of (pro-)inflammation and oxidative stress reactions and compared with the reference material carbon black. Single exposure to all tested GRM at the two different exposure concentrations (∼300 and 1000 ng/cm2) did not initiate an observable adverse effect to the 3D lung model under acute exposure scenarios.

Aim: Design nanostructured lipid carriers (NLC) to facilitate drug delivery to tuberculosis-infected areas, exploiting macrophage mannose receptors and assess their uptake in a 3D human lung model. Materials & methods: NLCs and mannosylated-NLCs were synthetized and characterized. Their uptake and biocompatibility were tested in a 3D human lung model. Results: The formulations have appropriate size (170–202 nm) and morphology for lung deposition. Cell membrane integrity was maintained and no significant pro-inflammatory cytokine (IL-1β, IL-8 and TNF-α) secretion or morphological changes were observed 24 h post nanoparticles exposure. NLCs and mannosylated NLCs were distributed in the apical side of the lung tissue, both in macrophages and in epithelial cells. Conclusion: NLCs are biocompatible carriers and can be used for pulmonary drug delivery.

Background Volcanic plumes are complex environments composed of gases and ash particles, where chemical and physical processes occur at different temperature and compositional regimes. Commonly, soluble sulphate- and chloride-bearing salts are formed on ash as gases interact with ash surfaces. Exposure to respirable volcanic ash following an eruption is potentially a significant health concern. The impact of such gas-ash interactions on ash toxicity is wholly un-investigated. Here, we study, for the first time, whether the interaction of volcanic particles with sulphur dioxide (SO2) gas, and the resulting presence of sulphate salt deposits on particle surfaces, influences toxicity to the respiratory system, using an advanced in vitro approach. Methods To emplace surface sulphate salts on particles, via replication of the physicochemical reactions that occur between pristine ash surfaces and volcanic gas, analogue substrates (powdered synthetic volcanic glass and natural pumice) were exposed to SO2 at 500 °C, in a novel Advanced Gas-Ash Reactor, resulting in salt-laden particles. The solubility of surface salt deposits was then assessed by leaching in water and geochemical modelling. A human multicellular lung model was exposed to aerosolised salt-laden and pristine (salt-free) particles, and incubated for 24 h. Cell cultures were subsequently assessed for biological endpoints, including cytotoxicity (lactate dehydrogenase release), oxidative stress (oxidative stress-related gene expression; heme oxygenase 1 and NAD(P)H dehydrogenase [quinone] 1) and its (pro-)inflammatory response (tumour necrosis factor α, interleukin 8 and interleukin 1β at gene and protein levels). Results In the lung cell model no significant effects were observed between the pristine and SO2-exposed particles, indicating that the surface salt deposits, and the underlying alterations to the substrate, do not cause acute adverse effects in vitro. Based on the leachate data, the majority of the sulphate salts from the ash surfaces are likely to dissolve in the lungs prior to cellular uptake. Conclusions The findings of this study indicate that interaction of volcanic ash with SO2 during ash generation and transport does not significantly affect the respiratory toxicity of volcanic ash in vitro. Therefore, sulphate salts are unlikely a dominant factor controlling variability in in vitro toxicity assessments observed during previous eruption response efforts.

Following our call to join in the discussion over the suitability of implementing a reporting checklist for bio–nano papers, the community responds.

International audience; The present work aims to study the effects that acute exposure to low concentrations of silver nanoparticles (AgNPs) cause in digestive glands of terrestrial isopods (Porcellio scaber). The experiments were designed to integrate different analytical techniques, such as transmission electron microscopy, atomic absorption spectroscopy, proton induced X-ray emission, and Fourier transform IR imaging (FTIRI), in order to gain a comprehensive insight into the process from the AgNPs’ synthesis to their interaction with biological tissues in vivo. To this aim, terrestrial isopods were fed with AgNPs having different shapes, sizes, and concentrations. For all the tested conditions, no toxicity at the whole organism level was observed after 14 days of exposure. However, FTIRI showed that AgNPs caused detectable local changes in proteins, lipids, nucleic acids and carbohydrates at the tissue level, to an extent dependent on the interplay of the AgNPs’ properties: shape, size, concentration and dissolution of ions from them.

Due to economic, practical, ethical, and scientific reasons, researchers, among others, are pushing for alternative in vitro test methods to replace or reduce existing animal experiments. In order for these tests to be more broadly used by the industrial sector and regulatory bodies, orchestrated efforts are required to show the robustness and reliability of in vitro methods, which can accelerate the use for early screening testing. Another way of increasing the use of alternatives is to coordinate validation studies, that is, multi-laboratory trials, and to gain regulatory approval and instatement as test guidelines or standard method. However, awareness of the exact standardization, validation, and approval process has been a major obstacle for many researchers. Herein, the process has been broken down into three main phases: i) test method development; ii) intra- and inter-laboratory validation; and iii) regulatory acceptance. This general process applies to all alternative methods seeking validation and approval, although the intricacies of different toxicological endpoints and/or chemical sectors may lead to additional work, particularly in the validation stage. The authors' aim is to provide insight in the development process of alternative methods with a focus on in vitro cell culture methods over validation to regulatory acceptance.

The presence of ascites in the peritoneal cavity leads to morphological and functional changes of the peritoneal mesothelial cell layer. Cells loose cell-cell interactions, rearrange their cytoskeleton, activate the production of fibronectin, and change their cell surface morphology in a proinflammatory environment. Moreover, ovarian cancer cell adhesion has been shown to be facilitated by these changes due to increased integrin- and CD44-mediated binding sites. In this study, the biological responsiveness of the human pleural mesothelial cell line MeT-5A to patient-derived and artificial ascites was studiedin vitroand adhesion of ovarian cancer cells, i.e. SKOV-3 cells, investigated. Changes were mainly observed in cells exposed to artificial ascites containing higher cytokine concentrations than patient-derived ascites. Interestingly, reduced cell-cell interactions were already observed in untreated MeT-5A cells and effects on tight junction protein expression and permeability upon exposure to ascites were minor. Ascites induced upregulation of CDC42 effector protein 2 expression, which affects stress fiber formation, however significant F-actin reorganization was not observed. Moreover, fibronectin production remained unchanged. Analysis of mesothelial cell surface characteristics showed upregulated expression of intercellular adhesion molecule 1, slightly increased hyaluronic acid secretion and decreased microvillus expression upon exposure to ascites. Nevertheless, the observed changes were not sufficient to facilitate adhesion of SKOV-3 cells on MeT-5A cell layer. This study revealed that MeT-5A cells show a reduced biological responsiveness to the presence of ascites, in contrast to published studies on primary human peritoneal mesothelial cells.

Inhalation of combustion-derived ultrafine particles (≤0.1 μm) has been found to be associated with pulmonary and cardiovascular diseases. However, correlation of the physicochemical properties of carbon-based particles such as surface charge and agglomeration state with adverse health effects has not yet been established, mainly due to limitations related to the detection of carbon particles in biological environments. The authors have therefore applied model particles as mimics of simplified particles derived from incomplete combustion, namely, carbon nanodots (CNDs) with different surface modifications and fluorescent properties. Their possible adverse cellular effects and their biodistribution pattern were assessed in a three-dimensional (3D) lung epithelial tissue model. Three different CNDs, namely, nitrogen, sulfur codoped CNDs (N,S-CNDs) and nitrogen doped CNDs (N-CNDs-1 and N-CNDs-2), were prepared by microwave-assisted hydrothermal carbonization using different precursors or different microwave systems. These CNDs were found to possess different chemical and photophysical properties. The surfaces of nanodots N-CNDs-1 and N-CNDs-2 were positively charged or neutral, respectively, arguably due to the presence of amine and amide groups, while the surfaces of N,S-CNDs were negatively charged, as they bear carboxylic groups in addition to amine and amide groups. Photophysical measurements showed that these three types of CNDs displayed strong photon absorption in the UV range. Both N-CNDs-1 and N,S-CNDs showed weak fluorescence emission, whereas N-CNDs-2 showed intense emission. A 3D human lung model composed of alveolar epithelial cells (A549 cell line) and two primary immune cells, i.e., macrophages and dendritic cells, was exposed to CNDs via a pseudo-air-liquid interface at a concentration of 100 μg/ml. Exposure to these particles for 24 h induced no harmful effect on the cells as assessed by cytotoxicity, cell layer integrity, cell morphology, oxidative stress, and proinflammatory cytokines release. The distribution of the CNDs in the lung model was estimated by measuring the fluorescence intensity in three different fractions, e.g., apical, intracellular, and basal, after 1, 4, and 24 h of incubation, whereby reliable results were only obtained for N-CNDs-2. It was shown that N-CNDs-2 translocate rapidly, i.e., >40% in the basal fraction within 1 h and almost 100% after 4 h, while ca. 80% of the N-CNDs-1 and N,S-CNDs were still located on the apical surface of the lung cells after 1 h. This could be attributed to the agglomeration behavior of N-CNDs-1 or N,S-CNDs. The surface properties of the N-CNDs bearing amino and amide groups likely induce greater uptake as N-CNDs could be detected intracellularly. This was less evident for N,S-CNDs, which bear carboxylic acid groups on their surface. In conclusion, CNDs have been designed as model systems for carbon-based particles; however, their small size and agglomeration behavior made their quantification by fluorescence measurement challenging. Nevertheless, it was demonstrated that the surface properties and agglomeration affected the biodistribution of the particles at the lung epithelial barrier in vitro. Inhalation of combustion-derived ultrafine particles (≤0.1 μm) has been found to be associated with pulmonary and cardiovascular diseases. However, correlation of the physicochemical properties of carbon-based particles such as surface charge and agglomeration state with adverse health effects has not yet been established, mainly due to limitations related to the detection of carbon particles in biological environments. The authors have therefore applied model particles as mimics of simplified particles derived from incomplete combustion, namely, carbon nanodots (CNDs) with different surface modifications and fluorescent properties. Their possible adverse cellular effects and their biodistribution pattern were assessed in a three-dimensional (3D) lung epithelial tissue model. Three different CNDs, namely, nitrogen, sulfur codoped CNDs (N,S-CNDs) and nitrogen doped CNDs (N-CNDs-1 and N-CND...

Communities resident in urban areas located near active volcanoes can experience volcanic ash exposures during, and following, an eruption, in addition to sustained exposures to high concentrations of anthropogenic air pollutants (e.g., vehicle exhaust emissions). Inhalation of anthropogenic pollution is known to cause the onset of, or exacerbate, respiratory and cardiovascular diseases. It is further postulated similar exposure to volcanic ash can also affect such disease states. Understanding of the impact of combined exposure of volcanic ash and anthropogenic pollution to human health, however, remains limited. The aim of this study was to assess the biological impact of combined exposure to respirable volcanic ash (from Soufrière Hills volcano (SHV), Montserrat and Chaitén volcano (ChV), Chile; representing different magmatic compositions and eruption styles) and freshly-generated complete exhaust from a gasoline vehicle. A multicellular human lung model (an epithelial cell-layer composed of A549 alveolar type II-like cells complemented with human blood monocyte-derived macrophages and dendritic cells cultured at the air-liquid interface) was exposed to diluted exhaust (1:10) continuously for 6 h, followed by immediate exposure to the ash as a dry powder (0.54 ± 0.19 μg/cm2 and 0.39 ± 0.09 μg/cm2 for SHV and ChV ash, respectively). After an 18 h incubation, cells were exposed again for 6 h to diluted exhaust, and a final 18 h incubation (at 37 °C and 5% CO2). Cell cultures were then assessed for cytotoxic, oxidative stress and (pro-)inflammatory responses. Results indicate that, at all tested (sub-lethal) concentrations, co-exposures with both ash samples induced no significant expression of genes associated with oxidative stress (HMOX1, NQO1) or production of (pro-)inflammatory markers (IL-1β, IL-8, TNF-α) at the gene and protein levels. In summary, considering the employed experimental conditions, combined exposure of volcanic ash and gasoline vehicle exhaust has a limited short-term biological impact to an advanced lung cell in vitro model.

The rapid development of engineered nanomaterials demands for a fast and reliable assessment of their health hazard potential. A plethora of experimental approaches have been developed and are widely employed in conventional toxicological approaches. However, the specific properties of nanomaterials such as smaller size but larger surface area, and high catalytic reactivity and distinctive optical properties compared to their respective bulk entities, often disable a straightforward use of established in vitro approaches. Herein, we provide an overview of the current state-of the art nanomaterial hazard assessment strategies using in vitro approaches. This perspective has been developed based on a thorough review of over 200 studies employing such methods to assess the biological response upon exposure to a diverse array of nanomaterials. The majority of the studies under review has been, but not limited to, engaged in the European 7th Framework Programme for Research and Technological Development and published in the last five years. Based on the most widely used methods and/or the most relevant biological endpoints, we have provided some general recommendations on the use of the selected approaches which would the most closely mimic realistic exposure scenarios as well as enabling to yield fast, reliable and reproducible data on the nanomaterial-cell response in vitro. In addition, the applicability of the approaches to translate in vitro outcomes to leverage those of in vivo studies has been proposed. It is finally suggested that an improved comprehension of the approaches with its limitations used for nanomaterials' hazard assessment in vitro will improve the interpretation of the existing nanotoxicological data as well as underline the basic principles in understanding interactions of engineered nanomaterials at a cellular level; this all is imperative for their safe-by- design strategies, and should also enable subsequent regulatory approvals.

Fullerenes (C60) are some of the most promising carbon nanomaterials to be used for medical applications as drug delivery agents. Computational and experimental studies have proposed their ability to enter cells by penetrating lipid bilayers. The aim of our study was to provide experimental evidence on whether pristine C60 in physiological media could penetrate cell membranes. The effect was tested on phospholipid vesicles (liposomes) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and validated on isolated human red blood cells (RBCs). We incubated the liposomes in an aqueous suspension of C60 and dissolved the lipids and C60 together in chloroform and subsequently formatted the liposomes. By differential scanning calorimetry measurements, we assessed the effect of C60 on the phospholipid thermal profile. The latter was not affected after the incubation of liposomes in the C60 suspension; also, a shape transformation of RBCs did not occur. Differently, by dispersing both C60 and the phospholipids in chloroform, we confirmed the possible interaction of C60 with the bilayer. We provide experimental data suggesting that the suspension medium is an important factor in determining the C60-membrane interaction, which is not always included in computational studies. Since the primary particle size is not the only crucial parameter in C60-membrane interactions, it is important to determine the most relevant characteristics of their effects on membranes.

As the range of engineered nanoparticles (NPs) designed as specific carriers increases, for example for cell targeting and drug delivery, the question on how many NPs are interacting or are taken up by cells is becoming increasingly important for any potential biomedical application. On one hand, the delivered dose of such NPs to the targeted cells is a key parameter in the assessment of their efficiency to perform the desired action (e.g., deliver the therapeutic substance or induce a specific effect), on the other hand, the assessment of intracellular NPs is crucial also from the safety aspect as NPs might come unintentionally in contact by untargeted cells. Particularly from the regulative perspective, it is important that reproducible and reliable analytical methods for the intracellular quantification of NPs are available at an early stage in the development in order to correlate the cell burden of NPs with their possible effects at a cellular level.

Carbon-based nanomaterials (NM) are promising candidates for a myriad of applications ranging from drug delivery to biosensing platforms. In the physiological environment, proteins can be adsorbed onto the surface of NM that can alter their structure and function. Little is known of the effect of NM on larger proteins and enzymes and an attempt has been made in this study to investigate the effect of carbon-based NM such as carbon black (CB), graphene oxide (GO) and fullerene (C60) on the adsorption and activity of acetylcholinesterase (AChE), a key enzyme present in brain, blood and nervous system and a suitable neurotoxicity biomarker. Experimental and computational results showed that all the carbon-based NM tested adsorb AChE but they have different effects on the catalytic activity of the enzyme. The most efficient AChE inhibitor is CB. In contrast, AChE adsorbed on the GO surface retains its native conformation and most of its activity. As compared to GO and CB, C60 was found to be an inefficient adsorbent of AChE. The distinctive adsorption pattern of NM and their inhibitory potential could be related to the surface characteristics of NM. Our studies also demonstrate the potential of GO as a substrate for immobilization of AChE.

The respiratory tract is in constant contact with inhaled antigens from the external environment. In order to shape its line of defense, it is populated by various types of immune cells. Taking into account the scientific breakthroughs of nanomedicine and nanoparticle drug delivery, we can think of the respiratory tract as an ideal target organ to study and develop nanocarrier-based vaccines to treat respiratory tract disorders. Nanoparticles have been proven capable of specific cell targeting and, when suitably engineered, are able to induce an immunomodulatory effect. The aim of this review is to highlight in vitro approaches to the study of nanoparticle-lung immune cell interactions and recent advances in the targeting of immune cells using nanoparticle-based systems.

There are some molecular dynamic simulations but a paucity of experimental evidence of the effects of C-60 fullerene on lipid bilayers. The aim of this study is to assess the potential for disruption of the lipid bilayer by C-60 suspended in water. We selected a C-60 suspension that has previously been shown to provoke cell membrane destabilisation in vivo. Electromobility measurements show significant negative surface charge on the C-60 nanoparticles suspended in a glucose solution and a zeta potential of −26 mV. The prevalent C-60 clusters have hydrodynamic radii of approximately 2 nm. Phase contrast microscopy and computer aided image analysis results show that C-60 causes shape transformations and rupture of unilamellar phospholipid vesicles, indicative of changes in their average mean curvature. Small-angle X-ray scattering reveals that C-60 provokes disruptions of external membranes of multilamellar vesicles only after freeze and thaw cycles. Here, the liposomes undergo breakage and annealing steps which increase the probability for fullerenes to insert into the MLVs. Our experimental findings confirm the potential of C-60 to reconstruct lipids in biological membranes. This research enhances our understanding of the impact of engineered nanoparticles on cell membranes.

Sterilization is the final surface treatment procedure of all implantable devices and is one of the key factors which have to be considered before implementation. Since different sterilization procedures for all implantable devices influence mechanical properties as well as biological response, the influence of different sterilization techniques on titanium nanotubes was studied. Commonly used sterilization techniques such as autoclaving, ultra-violet light sterilization, hydrogen peroxide plasma sterilization as well as the not so frequently used gaseous oxygen plasma sterilization were used. Three different nanotube diameters; 15 nm, 50 nm and 100 nm were employed to study the effects of various sterilization techniques. It was observed that autoclave sterilization resulted in destruction of nanotubular features on all three studied nanotube diameters, while UV-light and both kinds of plasma sterilization did not cause any significant morphological changes on the surfaces. Differences between the sterilization techniques employed influenced cytocompatibility, especially in the case of nanotubes with 100 nm diameter.

Poornima Budime Santhosh,1,* Barbara Drašler,2,* Damjana Drobne,2 Mateja Erdani Kreft,3 Slavko Kralj,4 Darko Makovec,4 Nataša Poklar Ulrih1,5 1Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, 2Department of Biology, Biotechnical Faculty, University of Ljubljana, 3Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, 4Department for Materials Synthesis, Jožef Stefan Institute, 5Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Ljubljana, Slovenia *These authors share equal first authorship Abstract: Superparamagnetic iron oxide nanoparticles (SPIONs) with multifunctional properties have shown great promise in theranostics. The aim of our work was to compare the effects of SPIONs on the fluidity and phase transition of the liposomal membranes prepared with zwitterionic phosphatidylcholine lipids. In order to study if the surface modification of SPIONs has any influence on these membrane properties, we have used four types of differently functionalized SPIONs, such as: plain SPIONs (primary size was shown to be ~11 nm), silica-coated SPIONs, SPIONs coated with silica and functionalized with positively charged amino groups or negatively charged carboxyl groups (the primary size of all the surface-modified SPIONs was ~20 nm). Small unilamellar vesicles prepared with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipids and multilamellar vesicles prepared with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine lipids were encapsulated or incubated with the plain and surface-modified SPIONs to determine the fluidity and phase transition temperature of the bilayer lipids, respectively. Fluorescent anisotropy and differential scanning calorimetric measurements of the liposomes that were either encapsulated or incubated with the suspension of SPIONs did not show a significant difference in the lipid ordering and fluidity; though the encapsulated SPIONs showed a slightly increased effect on the fluidity of the model membranes in comparison with the incubated SPIONs. This indicates the low potential of the SPIONs to interact with the nontargeted cell membranes, which is a desirable factor for in vivo applications. Keywords: encapsulated SPIONs, incubated SPIONs, zwitterionic liposomes, membrane integrity, phase behavior

The applications of zinc oxide (ZnO) nanowires (NWs) in implantable wireless devices, such as diagnostic nanobiosensors and nanobiogenerators, have recently attracted enormous attention due to their unique properties. However, for these implantable nanodevices, the biocompatibility and the ability to control the behaviour of cells in contact with ZnO NWs are demanded for the success of these implantable devices, but to date, only a few contrasting results from their biocompatibility can be found. There is a need for more research about the biocompatibility of ZnO nanostructures and the adhesion and viability of cells on the surface of ZnO nanostructures. Here, we introduce synthesis of a new nature-inspired nanostructured ZnO urchin, with the dimensions of the ZnO urchin’s acicula being controllable. To examine the biocompatibility and behaviour of cells in contact with the ZnO urchin, the Madin-Darby canine kidney (MDCK) epithelial cell line was chosen as an in vitro experimental model. The results of the viability assay indicated that, compared to control, the number of viable cells attached to the surface of the ZnO urchin and its surrounding area were reduced. The measurements of the Zn contents of cell media confirmed ZnO dissolution, which suggests that the ZnO dissolution in cell culture medium could lead to cytotoxicity. A purposeful reduction of ZnO cytotoxicity was achieved by surface coating of the ZnO urchin with poly(vinylidene fluorid-co-hexafluoropropylene) (PVDF-HFP), which changed the material matrix to slow the Zn ion release and consequently reduce the cytotoxicity of the ZnO urchin without reducing its functionality. Electronic supplementary material The online version of this article (doi:10.1186/s11671-015-1145-3) contains supplementary material, which is available to authorized users.

The coupling of fullerene (C60) to the structure and elasticity of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers has been explored by synchrotron small angle X-ray scattering. Multilamellar vesicles were loaded with 0, 2 and 10 mol.% of C60 and studied in a temperature range from 15 to 65 °C. The addition of C60 caused an increase in the bilayer undulations (∼20%), in the bilayer separation (∼15%), in the linear expansion coefficient and caused a drop in the bending rigidity of the bilayers (20–40%). Possible damaging effects of fullerene on biomembranes are mainly discussed on the basis of altered bilayer fluidity and elasticity changes.

The identification of biomaterials that support appropriate cellular attachment, proliferation, and functions is critical for tissue engineering and cell therapy. There is a growing interest in functional organic/inorganic composites where a small amount of nanometer-sized material yields better physicochemical properties for cells to attach, grow, and differentiate. In this work, we prepared polymer/nanodiamond composite layers based on hexamethyldisiloxane and detonation-generated nanodiamond (DND) particles, in which the particles were either embedded into a polymer matrix or deposited on the preliminary formed plasma-polymerized (PP) layer. The surface properties of composites, such as roughness and wettability, as well as adhesion, growth, and functions of osteosarcoma MG-63 cells and primary rat mesenchymal stem cells were studied. We aimed to investigate the influence of the incorporation methods of DND into the polymer on the material surface properties and the cell response in order to control them by manipulating diamond-containing composite surfaces. We found differences between both composites in respect to their physicochemical properties and to the cell behavior suggesting that the method of particle incorporation into polymers should be taken in account during the development of new biomaterials for a specific application.

The aim of this study was to determine whether citric acid adsorbed onto cobalt ferrite (CoFe2O4) nanoparticles (NPs) influences the bioavailability of their constituents Co and Fe. Dissolution of Co and Fe was assessed by two measures: (i) in aqueous suspension using chemical analysis, prior to application onto the food of test organisms; and (ii) in vivo, measuring the bioavailability in the model terrestrial invertebrate (Porcellio scaber, Isopoda, Crustacea). The isopods were exposed to citric-acid-adsorbed CoFe2O4 NPs for 2 weeks, and tissue accumulation of Co and Fe was assessed. This was compared to pristine CoFe2O4 NPs, and CoCl2 and Fe(III) salts as positive controls. The combined data shows that citric acid enhances free metal ion concentration from CoFe2O4 NPs in aqueous suspension, although in vivo, very similar amounts of assimilated Co were found in isopods exposed to both types of NPs. Therefore, evaluation of the dissolution in suspension by chemical means is not a good predictor of metal assimilation of this model organism; body assimilation of Co and Fe is rather governed by the physiological capacity of P. scaber for the uptake of these metals. Moreover, we propose that citric acid, due to its chelating properties, may hinder the uptake of Co that dissolves from citric-acid-adsorbed CoFe2O4 NPs, if citric acid is present in sufficient quantity.

Barbara Drašler,1 Damjana Drobne,1–3 Sara Novak,1 Janez Valant,1 Sabina Boljte,1,4 Lado Otrin,1 Michael Rappolt,5,6 Barbara Sartori,5 Aleš Iglic,7 Veronika Kralj-Iglic,8 Vid Šuštar,9 Darko Makovec,3,10 Sašo Gyergyek,10 Matej Hocevar,11 Matjaž Godec,11 Jernej Zupanc11University of Ljubljana, Biotechnical Faculty, Department of Biology, Ljubljana, 2Centre of Excellence in Advanced Materials and Technologies for the Future, Ljubljana, 3Centre of Excellence in Nanoscience and Nanotechnology, Ljubljana, 4Institute of Microbial Sciences and Technologies, Ljubljana, Slovenia; 5Institute of Inorganic Chemistry, Graz University of Technology, Basovizza, Italy; 6School of Food Science and Nutrition, University of Leeds, Leeds, UK; 7Faculty of Electrical Engineering, University of Ljubljana, Ljubljana, 8Faculty of Health Sciences, Laboratory of Clinical Biophysics, University of Ljubljana, Ljubljana 9Laboratory of Clinical Biophysics, Chair of Orthopaedics, Faculty of Medicine, University of Ljubljana, Ljubljana, 10Institute Jožef Stefan, Ljubljana, 11Institute of Metals and Technology, Ljubljana, SloveniaBackground: The purpose of this work is to provide experimental evidence on the interactions of suspended nanoparticles with artificial or biological membranes and to assess the possibility of suspended nanoparticles interacting with the lipid component of biological membranes.Methods: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid vesicles and human red blood cells were incubated in suspensions of magnetic bare cobalt ferrite (CoFe2O4) or citric acid (CA)-adsorbed CoFe2O4 nanoparticles dispersed in phosphate-buffered saline and glucose solution. The stability of POPC giant unilamellar vesicles after incubation in the tested nanoparticle suspensions was assessed by phase-contrast light microscopy and analyzed with computer-aided imaging. Structural changes in the POPC multilamellar vesicles were assessed by small angle X-ray scattering, and the shape transformation of red blood cells after incubation in tested suspensions of nanoparticles was observed using scanning electron microscopy and sedimentation, agglutination, and hemolysis assays.Results: Artificial lipid membranes were disturbed more by CA-adsorbed CoFe2O4 nanoparticle suspensions than by bare CoFe2O4 nanoparticle suspensions. CA-adsorbed CoFe2O4-CA nanoparticles caused more significant shape transformation in red blood cells than bare CoFe2O4 nanoparticles.Conclusion: Consistent with their smaller sized agglomerates, CA-adsorbed CoFe2O4 nanoparticles demonstrate more pronounced effects on artificial and biological membranes. Larger agglomerates of nanoparticles were confirmed to be reactive against lipid membranes and thus not acceptable for use with red blood cells. This finding is significant with respect to the efficient and safe application of nanoparticles as medicinal agents.Keywords: CoFe2O4, nanoparticles, agglomerates, human red blood cells, lipid vesicles

We present a method for giant lipid vesicle shape analysis that combines manually guided large-scale video microscopy and computer vision algorithms to enable analyzing vesicle populations. The method retains the benefits of light microscopy and enables non-destructive analysis of vesicles from suspensions containing up to several thousands of lipid vesicles (1-50 µm in diameter). For each sample, image analysis was employed to extract data on vesicle quantity and size distributions of their projected diameters and isoperimetric quotients (measure of contour roundness). This process enables a comparison of samples from the same population over time, or the comparison of a treated population to a control. Although vesicles in suspensions are heterogeneous in sizes and shapes and have distinctively non-homogeneous distribution throughout the suspension, this method allows for the capture and analysis of repeatable vesicle samples that are representative of the population inspected.

In the present study the biological response to various nanotopographic features after gaseous plasma treatment were studied. The usefulness of nanostructured surfaces for implantable materials has already been acknowledged, while less is known on the combined effect of nanostructured plasma modified surfaces. In the present work the influence of oxygen plasma treatment on nanostructured titanium oxide (TiO2) surfaces was studied. Characterization of the TiO2 surface chemical composition and morphological features was analyzed after plasma modification by x-ray photoelectron spectroscopy and by scanning electron microscopy while surface wettability was studied with measuring the water contact angle. Cell adhesion and morphology was assessed from images taken with scanning electron microscopy, whereas cell viability was measured with a calorimetric assay. The obtained results showed that oxygen plasma treatment of TiO2 nanotube surfaces significantly influences the adhesion and morphology of osteoblast-like cells in comparison to untreated nanostructured surfaces. Marked changes in surface composition of plasma treated surfaces were observed, as plasma treatment removed hydrocarbon contamination and removed fluorine impurities, which were present due to the electrochemical anodization process. However no differences in wettability of untreated and plasma treated surfaces were noticed. Treatment with oxygen plasma stimulated osteoblast-like cell adhesion and spreading on the nanostructured surface, suggesting the possible use of oxygen plasma surface treatment to enhance osteoblast-like cell response.

Background We 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. Results We 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. Conclusions CB 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.

Background Massive 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. Results Washed 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 Conclusions These results indicate that TiO2 and ZnO EPs cause erythrocyte aggregation and could be potentially prothrombogenic, while ZnO could also cause membrane rupture.