Recovering What Matters: High Protein Recovery after Endotoxin Removal from LPS-Contaminated Formulations Using Novel Anti-Lipid A Antibody Microparticle Conjugates.

Endotoxins or lipopolysaccharides (LPS), found in the outer membrane of Gram-negative bacterial cell walls, can stimulate the human innate immune system, leading to life-threatening symptoms. Therefore, regulatory limits for endotoxin content apply to injectable pharmaceuticals, and excess LPS must be removed before commercialization. The majority of available endotoxin removal systems are based on the non-specific adsorption of LPS to charged and/or hydrophobic surfaces. Albeit effective to remove endotoxins, the lack of specificity can result in the unwanted loss of essential proteins from the pharmaceutical formulation. In this work, we developed microparticles conjugated to anti-Lipid A antibodies for selective endotoxin removal. Anti-Lipid A particles were characterized using flow cytometry and microscopy techniques. These particles exhibited a depletion capacity > 6 ×103 endotoxin units/mg particles from water, as determined with two independent methods (Limulus Amebocyte Lysate test and nanoparticle tracking analysis). Additionally, we compared these particles with a non-specific endotoxin removal system in a series of formulations of increasing complexity: bovine serum albumin in water < insulin in buffer < birch pollen extracts. We demonstrated that the specific anti-Lipid A particles show a higher protein recovery without compromising their endotoxin removal capacity. Consequently, we believe that the specificity layer integrated by the anti-Lipid A antibody could be advantageous to enhance product yield.

Role of Common Cell Culture Media Supplements on Citrate-Stabilized Gold Nanoparticle Protein Corona Formation, Aggregation State, and the Consequent Impact on Cellular Uptake.

Sodium citrate-stabilized gold nanoparticles (AuNPs) are destabilized when dispersed in cell culture media (CCMs). This may promote their aggregation and subsequent sedimentation, or under the proper conditions, their interaction with dispersed proteins can lead to the formation of a NP-stabilizing protein corona. CCMs are ionic solutions that contain growth substances which are typically supplemented, in addition to serum, with different substances such as dyes, antioxidants, and antibiotics. In this study, the impact of phenol red, penicillin-streptomycin, l-glutamine, and ß-mercaptoethanol on the formation of the NP-protein corona in CCMs was investigated. Similar protein coronas were obtained except in the presence of antibiotics. Under these conditions, the protein corona took more time to be formed, and its density and composition were altered, as indicated by UV-vis spectroscopy, Z potential, dynamic light scattering, and liquid chromatography-mass spectrometry analyses. As a consequence of these modifications, a significantly different AuNP cellular uptake was measured, showing that NP uptake increased as did the NP aggregate formation. AuNP uptake studies performed in the presence of clathrin- and caveolin-mediated endocytosis inhibitors showed that neither clathrin receptors nor lipid rafts were significantly involved in the internalization mechanism. These results suggest that in these conditions, NP aggregation is the main mechanism responsible for their cellular uptake.

Nanoparticles and innate immunity: new perspectives on host defence.

The innate immune system provides the first line of defence against foreign microbes and particulate materials. Engineered nanoparticles can interact with the immune system in many different ways. Nanoparticles may thus elicit inflammation with engagement of neutrophils, macrophages and other effector cells; however, it is important to distinguish between acute and chronic inflammation in order to identify the potential hazards of nanoparticles for human health. Nanoparticles may also interact with and become internalised by dendritic cells, key antigen-presenting cells of the immune system, where a better understanding of these processes could pave the way for improved vaccination strategies. Nanoparticle characteristics such as size, shape and deformability also influence nanoparticle uptake by a plethora of immune cells and subsequent immune responses. Furthermore, the corona of adsorbed biomolecules on nanoparticle surfaces should not be neglected. Complement activation represents a special case of regulated and dynamic corona formation on nanoparticles with important implications in clearance and safety. Additionally, the inadvertent binding of bacterial lipopolysaccharide to nanoparticles is important to consider as this may skew the outcome and interpretation of immunotoxicological studies. Here, we discuss nanoparticle interactions with different cell types and soluble mediators belonging to the innate immune system.

Structural Alterations of Antigens at the Material Interface: An Early Decision Toolbox Facilitating Safe-by-Design Nanovaccine Development.

Nanomaterials have found extensive interest in the development of novel vaccines, as adjuvants and/or carriers in vaccination platforms. Conjugation of protein antigens at the particle surface by non-covalent adsorption is the most widely used approach in licensed particulate vaccines. Hence, it is essential to understand proteins' structural integrity at the material interface in order to develop safe-by-design nanovaccines. In this study, we utilized two model proteins, the wild-type allergen Bet v 1 and its hypoallergenic fold variant (BM4), to compare SiO2 nanoparticles with Alhydrogel® as particulate systems. A set of biophysical and functional assays including circular dichroism spectroscopy and proteolytic degradation was used to examine the antigens' structural integrity at the material interface. Conjugation of both biomolecules to the particulate systems decreased their proteolytic stability. However, we observed qualitative and quantitative differences in antigen processing concomitant with differences in their fold stability. These changes further led to an alteration in IgE epitope recognition. Here, we propose a toolbox of biophysical and functional in vitro assays for the suitability assessment of nanomaterials in the early stages of vaccine development. These tools will aid in safe-by-design innovations and allow fine-tuning the properties of nanoparticle candidates to shape a specific immune response.

When Would Immunologists Consider a Nanomaterial to be Safe? Recommendations for Planning Studies on Nanosafety.

The immune system is professional in recognizing and responding to non-self, including nanomaterials. Immune responses by professional and nonprofessional immune cells are thus nearly inevitable upon exposure of cells and organisms to such materials. The state of research into taking the immune system into account in nanosafety studies is reviewed and three aspects in which further improvements are desirable are identified: 1) Due to technical limitations, more stringent testing for endotoxin contamination should be made. 2) Since under overdose conditions immunity shows unphysiological responses, all doses used should be justified by being equivalent to tissue-delivered doses. 3) When markers of acute inflammation or cell stress are observed, functional assays are necessary to distinguish between homeostatic fluctuation and genuine defensive or tolerogenic responses. Since immune activation can also indicate that the immune system considers a stimulus to be harmless and induces tolerance, activation markers by themselves do not necessarily imply a danger to the body. Guidelines such as these are necessary to approach the point where specific nanomaterials are classified as safe based on reliable testing strategies.

Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species.

The interaction of a living organism with external foreign agents is a central issue for its survival and adaptation to the environment. Nanosafety should be considered within this perspective, and it should be examined that how different organisms interact with engineered nanomaterials (NM) by either mounting a defensive response or by physiologically adapting to them. Herein, the interaction of NM with one of the major biological systems deputed to recognition of and response to foreign challenges, i.e., the immune system, is specifically addressed. The main focus is innate immunity, the only type of immunity in plants, invertebrates, and lower vertebrates, and that coexists with adaptive immunity in higher vertebrates. Because of their presence in the majority of eukaryotic living organisms, innate immune responses can be viewed in a comparative context. In the majority of cases, the interaction of NM with living organisms results in innate immune reactions that eliminate the possible danger with mechanisms that do not lead to damage. While in some cases such interaction may lead to pathological consequences, in some other cases beneficial effects can be identified.

TGFß1 mimetic peptide modulates immune response to grass pollen allergens in mice.

BACKGROUND: Transforming growth factor ß1 (TGFß1) is a cytokine that exerts immunosuppressive functions, as reflected by its ability to induce regulatory T (Treg) cell differentiation and inhibit Th1 and Th2 responses. Hence, peptides that mimic the active core domain of TGFß1 may be promising candidates for modulation of the allergic response. This study aimed to investigate a synthetic TGFß1 mimetic peptide (TGFß1-mim) for its ability to modulate the immune response during allergic sensitization to grass pollen allergens. METHODS: The in vitro action of TGFß1-mim was evaluated in human lung epithelial cells, Jurkat cells, and rat basophilic leukemia cells. The in vivo action was evaluated in a murine model of Phl p 5 allergic sensitization. Additionally, the Th2 modulatory response was evaluated in IL-4 reporter mice. RESULTS: In vitro, TGFß1-mim downregulated TNF-α production, IL-8 gene expression, and cytokine secretion, upregulated IL-10 secretion, and inhibited Phl p 5-induced basophil degranulation. During Phl p 5 sensitization in mice, TGFß1-mim downregulated IL-2, IL-4, IL-5, IL-13, and IFN-γ, upregulated IL-10, and induced Treg cell production. Furthermore, mice treated with TGFß1-mim had lower levels of IgE, IgG1, IgG2a and higher levels of IgA antibodies than control mice. In a reporter mouse, the mimetic inhibited Th2 polarization. CONCLUSION: The TGFß1-mim efficiently modulated various important events that exacerbate the allergic microenvironment, including the production of main cytokines that promote Th1 and Th2 differentiation, and the induction of allergen-specific regulatory T cells, highlighting its potential use in therapeutic approaches to modulate the immune response toward environmental allergens.

Nitrated food proteins induce a regulatory immune response associated with allergy prevention after oral exposure in a Balb/c mouse food allergy model.

BACKGROUND: Food allergy is associated with a high personal health and economic burden. For immunomodulation toward tolerance, food compounds could be chemically modified, for example, by posttranslational protein nitration, which also occurs via diet-derived nitrating agents in the gastrointestinal tract. OBJECTIVE: We sought to analyze the effect of pretreatment with nitrated food proteins on the immune response in a mouse food allergy model and on human monocyte-derived dendritic cells (moDCs) and PBMCs. METHODS: The model allergen ovalbumin (OVA) was nitrated in different nitration degrees, and the secondary structures of proteins were determined by circular dichroism (CD). Allergy-preventive treatment with OVA, nitrated OVA (nOVA), and maximally nitrated OVA (nOVAmax) were performed before mice were immunized with or without gastric acid-suppression medication. Antibody levels, regulatory T-cell (Treg) numbers, and cytokine levels were evaluated. Human moDCs or PBMCs were incubated with proteins and evaluated for expression of surface markers, cytokine production, and proliferation of Th2 as well as Tregs. RESULTS: In contrast to OVA and nOVA, the conformation of nOVAmax was substantially changed. nOVAmax pretreated mice had decreased IgE as well as IgG1 and IgG2a levels and Treg numbers were significantly elevated, while cytokine levels remained at baseline level. nOVAmax induced a regulatory DC phenotype evidenced by a decrease of the activation marker CD86 and an increase in IL-10 production and was associated with a higher proliferation of memory Tregs. CONCLUSION: Oral pretreatment with highly nitrated proteins induces a tolerogenic immune response in the food allergy model and in human immune cells.

Interactions of TiO2 Nanoparticles with Ingredients from Modern Lifestyle Products and Their Effects on Human Skin Cells.

The number of consumer products containing nanoparticles (NPs) experienced a rapid increase during the past decades. However, most studies of nanosafety have been conducted using only pure NPs produced in the laboratory, while the interactions with other ingredients in consumer products have rarely been considered so far. In the present study, we investigated such interactions-with a special focus on modern lifestyle products (MLPs) used by adolescents. An extensive survey was undertaken at different high schools all over Austria to identify MLPs that either contain NPs or that could come easily in contact with NPs from other consumer products (such as TiO2 from sunscreens). Based on the results from a survey among secondary schools students, we focused on ingredients from Henna tattoos (2-hydroxy-1,4-naphtoquinone, HNQ, and p-phenylenediamine, PPD), fragrances (butylphenyl methylpropional, known as Lilial), cosmetics and skin-care products (four different parabens). As a cellular model, we decided to use neonatal normal human dermal fibroblasts (nNHDF), since skin contact is the main route of exposure for these compounds. TiO2 NPs interacted with these compounds as evidenced by alterations in their hydrodynamic diameter observed by nanoparticle tracking analysis. Combinations of TiO2 NPs with the different MLP components did not show altered cytotoxicity profiles compared to MLP components without TiO2 NPs. Nevertheless, altered cellular glutathione contents were detected after incubation of the cells with Lilial. This effect was independent of the presence of TiO2 NPs. Testing mixtures of NPs with other compounds from consumer products is an important approach to achieve a more reliable safety assessment.

The Impact of Nanoparticles on Innate Immune Activation by Live Bacteria.

The innate immune system evolved to detect and react against potential dangers such as bacteria, viruses, and environmental particles. The advent of modern technology has exposed innate immune cells, such as monocytes, macrophages, and dendritic cells, to a relatively novel type of particulate matter, i.e., engineered nanoparticles. Nanoparticles are not inherently pathogenic, and yet cases have been described in which specific nanoparticle types can either induce innate/inflammatory responses or modulate the activity of activated innate cells. Many of these studies rely upon activation by agonists of toll-like receptors, such as lipopolysaccharide or peptidoglycan, instead of the more realistic stimulation by whole live organisms. In this review we examine and discuss the effects of nanoparticles on innate immune cells activated by live bacteria. We focus in particular on how nanoparticles may interfere with bacterial processes in the context of innate activation, and confine our scope to the effects due to particles themselves, rather than to molecules adsorbed on the particle surface. Finally, we examine the long-lasting consequences of coexposure to nanoparticles and bacteria, in terms of potential microbiome alterations and innate immune memory, and address nanoparticle-based vaccine strategies against bacterial infection.

Particle toxicology and health - where are we?

BACKGROUND: Particles and fibres affect human health as a function of their properties such as chemical composition, size and shape but also depending on complex interactions in an organism that occur at various levels between particle uptake and target organ responses. While particulate pollution is one of the leading contributors to the global burden of disease, particles are also increasingly used for medical purposes. Over the past decades we have gained considerable experience in how particle properties and particle-bio interactions are linked to human health. This insight is useful for improved risk management in the case of unwanted health effects but also for developing novel medical therapies. The concepts that help us better understand particles' and fibres' risks include the fate of particles in the body; exposure, dosimetry and dose-metrics and the 5 Bs: bioavailability, biopersistence, bioprocessing, biomodification and bioclearance of (nano)particles. This includes the role of the biomolecule corona, immunity and systemic responses, non-specific effects in the lungs and other body parts, particle effects and the developing body, and the link from the natural environment to human health. The importance of these different concepts for the human health risk depends not only on the properties of the particles and fibres, but is also strongly influenced by production, use and disposal scenarios. CONCLUSIONS: Lessons learned from the past can prove helpful for the future of the field, notably for understanding novel particles and fibres and for defining appropriate risk management and governance approaches.

Correction to: Particle toxicology and health - where are we?

After the publication of this article [1] it was hihglighted that the number of deaths related to natural disasters was incorrectly reported in the second paragraph of the Hazards from Natural particulates and the evolution of the biosphere section. This correction article shows the correct and incorrect statement. This correction does not change the idea presented in the article that from an evolutionary view point, natural disasters account only for a small fraction of the people on the planet. The original article has been updated.

The protein corona suppresses the cytotoxic and pro-inflammatory response in lung epithelial cells and macrophages upon exposure to nanosilica.

Engineered amorphous silica nanoparticles (nanosilica) are one of the most abundant nanomaterials and are widely used in industry. Furthermore, novel nanosilica materials are promising theranostic tools for biomedicine. However, hazardous effects of nanosilica especially after inhalation into the lung have been documented. Therefore, the safe development of nanosilica materials urgently requires predictive assays to monitor toxicity. Here, we further investigate the impact of the protein corona on the biological activity of two different types of nanosilica (colloidal and pyrogenic) in lung cells. As previously described, adsorption of serum proteins to the nanosilica surface suppresses cytotoxicity in macrophages and lung epithelial cells. As the increase of pro-inflammatory mediators is a hallmark of inflammation in the lung upon nanosilica exposure, we studied the potential coupling of the cytotoxic and pro-inflammatory response in A549 human lung epithelial cells and RAW264.7 mouse macrophages. Indeed, cytotoxicity precedes the onset of pro-inflammatory gene expression and cytokine release as exemplified for IL-8 in A549 cells and TNF-alpha in RAW264.7 macrophages after exposure to 0-100 µg/mL nanosilica in medium without serum. Formation of a protein corona not only inhibited cellular toxicity, but also the pro-inflammatory response. Of note, uptake of nanosilica into cells was negligible in the absence, but enhanced in the presence of a protein corona. Hence, the prevailing explanation that the protein corona simply interferes with cellular uptake thus preventing adverse effects needs to be revisited. In conclusion, for the reliable prediction of adverse effects of nanosilica in the lung, in vitro assays should be performed in media not complemented with complete serum. However, in case of different exposure routes, e.g., injection into the blood stream as intended for biomedicine, the protein corona prevents acute toxic actions of nanosilica.

The role of B7 costimulation in benzene immunotoxicity and its potential association with cancer risk.

Benzene is a recognized human carcinogen; however, there are still some gaps in the knowledge regarding the mechanism of toxicity of this organic solvent and potential early biomarkers for the damage caused by it. In a previous study, our research group demonstrated that the adhesion molecules of the immune system (B7.1 and B7.2) could be potential biomarkers in the early detection of immunotoxicity caused by benzene exposure. Therefore, this study was developed to deepen the understanding regarding this important topic, aiming to contribute to the comprehension of the benzene toxicity mechanism mediated by B7.1 and B7.2 and its potential association with the risk of carcinogenicity. B7.1 and B7.2 protein expression in blood monocytes and B7.1 and B7.2 gene expression in PBMCs were evaluated. Additionally, complement C3 and C4 levels in serum were measured, as well as p53 gene expression in PBMCs. Seventy-four gas station workers (GSW group) and 71 non-occupationally exposed subjects (NEG) were evaluated. Our results demonstrated decreased levels of B7.1 and B7.2 protein and gene expression in the GSW group compared to the NEG (n = 71) (p < 0.01). Along the same lines, decreased levels of the complement system were observed in the GSW group (p < 0.01), demonstrating the impairment of this immune system pathway as well. Additionally, a reduction was observed in p53 gene expression in the GSA group (p < 0.01). These alterations were associated with both the benzene exposure biomarker evaluated, urinary trans, trans-muconic acid, and with exposure time (p < 0.05). Moreover, strong correlations were observed between the gene expression of p53 vs. B7.1 (r = 0.830; p < 0.001), p53 vs. B7.2 (r = 0.685; p < 0.001), and B7.1 vs. B7.2 (r = 0.702; p < 0.001). Taken together, these results demonstrate that the immune system co-stimulatory molecule pathway is affected by benzene exposure. Also, the decrease in p53 gene expression, even at low exposure levels, reinforces the carcinogenicity effect of benzene in this pathway. Therefore, our results suggest that the promotion of immune evasion together with a decrease in p53 gene expression may play an important role in the benzene toxicity mechanism. However, further and targeted studies are needed to confirm this proposition.

Bovine Serum Albumin Adsorption on TiO2 Colloids: The Effect of Particle Agglomeration and Surface Composition.

Protein adsorption at nanostructured oxides strongly depends on the synthesis conditions and sample history of the material investigated. We measured the adsorption of bovine serum albumin (BSA) to commercial Aeroxide TiO2 P25 nanoparticles in aqueous dispersions. Significant changes in the adsorption capacity were induced by mild sample washing procedures and attributed to the structural modification of adsorbed water and surface hydroxyls. Motivated by the lack of information about the sample history of commercial TiO2 nanoparticle samples, we used vapor-phase-grown TiO2 nanoparticles, a well-established model system for adsorption and photocatalysis studies, and performed on this material for the first time a systematic and quantitative BSA adsorption study. After alternating vacuum and oxygen treatment of the nanoparticle powders at elevated temperatures for surface purification, we determined size distributions covering both the size of the individualized nanoparticles and nanoparticle agglomerates using transmission electron microscopy (TEM), X-ray diffraction (XRD), and dynamic light scattering (DLS) in an aqueous dispersion. Quantitative BSA adsorption measurements at different pH values and thus variable combinations of surface-charged proteins and TiO2 nanoparticles revealed a consistent picture: BSA adsorbs only at the outer agglomerate surfaces without penetrating the interior of the agglomerates. This process levels at coverages of single monolayers, which resist consecutive simple washing procedures. A detailed analysis of the protein-specific IR amide bands reveals that the adsorption-induced protein conformational change is associated with a decrease in the helical content. This study underlines that robust qualitative and quantitative statements about protein adsorption and corona formation require well-documented and controllable surface properties of the nanomaterials involved.

A Novel Exposure System Termed NAVETTA for In Vitro Laminar Flow Electrodeposition of Nanoaerosol and Evaluation of Immune Effects in Human Lung Reporter Cells.

A new prototype air-liquid interface (ALI) exposure system, a flatbed aerosol exposure chamber termed NAVETTA, was developed to investigate deposition of engineered nanoparticles (NPs) on cultured human lung A549 cells directly from the gas phase. This device mimics human lung cell exposure to NPs due to a low horizontal gas flow combined with cells exposed at the ALI. Electrostatic field assistance is applied to improve NP deposition efficiency. As proof-of-principle, cell viability and immune responses after short-term exposure to nanocopper oxide (CuO)-aerosol were determined. We found that, due to the laminar aerosol flow and a specific orientation of inverted transwells, much higher deposition rates were obtained compared to the normal ALI setup. Cellular responses were monitored with postexposure incubation in submerged conditions, revealing CuO dissolution in a concentration-dependent manner. Cytotoxicity was the result of ionic and nonionic Cu fractions. Using the optimized inverted ALI/postincubation procedure, pro-inflammatory immune responses, in terms of interleukin (IL)-8 promoter and nuclear factor kappa B (NFκB) activity, were observed within short time, i.e. One hour exposure to ALI-deposited CuO-NPs and 2.5 h postincubation. NAVETTA is a novel option for mimicking human lung cell exposure to NPs, complementing existing ALI systems.

Air Pollution and Climate Change Effects on Allergies in the Anthropocene: Abundance, Interaction, and Modification of Allergens and Adjuvants.

Air pollution and climate change are potential drivers for the increasing burden of allergic diseases. The molecular mechanisms by which air pollutants and climate parameters may influence allergic diseases, however, are complex and elusive. This article provides an overview of physical, chemical and biological interactions between air pollution, climate change, allergens, adjuvants and the immune system, addressing how these interactions may promote the development of allergies. We reviewed and synthesized key findings from atmospheric, climate, and biomedical research. The current state of knowledge, open questions, and future research perspectives are outlined and discussed. The Anthropocene, as the present era of globally pervasive anthropogenic influence on planet Earth and, thus, on the human environment, is characterized by a strong increase of carbon dioxide, ozone, nitrogen oxides, and combustion- or traffic-related particulate matter in the atmosphere. These environmental factors can enhance the abundance and induce chemical modifications of allergens, increase oxidative stress in the human body, and skew the immune system toward allergic reactions. In particular, air pollutants can act as adjuvants and alter the immunogenicity of allergenic proteins, while climate change affects the atmospheric abundance and human exposure to bioaerosols and aeroallergens. To fully understand and effectively mitigate the adverse effects of air pollution and climate change on allergic diseases, several challenges remain to be resolved. Among these are the identification and quantification of immunochemical reaction pathways involving allergens and adjuvants under relevant environmental and physiological conditions.

Enzyme adsorption-induced activity changes: a quantitative study on TiO2 model agglomerates.

BACKGROUND: Activity retention upon enzyme adsorption on inorganic nanostructures depends on different system parameters such as structure and composition of the support, composition of the medium as well as enzyme loading. Qualitative and quantitative characterization work, which aims at an elucidation of the microscopic details governing enzymatic activity, requires well-defined model systems. RESULTS: Vapor phase-grown and thermally processed anatase TiO2 nanoparticle powders were transformed into aqueous particle dispersions and characterized by dynamic light scattering and laser Doppler electrophoresis. Addition of ß-galactosidase (ß-gal) to these dispersions leads to complete enzyme adsorption and the generation of ß-gal/TiO2 heteroaggregates. For low enzyme loadings (~4% of the theoretical monolayer coverage) we observed a dramatic activity loss in enzymatic activity by a factor of 60-100 in comparison to that of the free enzyme in solution. Parallel ATR-IR-spectroscopic characterization of ß-gal/TiO2 heteroaggregates reveals an adsorption-induced decrease of the ß-sheet content and the formation of random structures leading to the deterioration of the active site. CONCLUSIONS: The study underlines that robust qualitative and quantitative statements about enzyme adsorption and activity retention require the use of model systems such as anatase TiO2 nanoparticle agglomerates featuring well-defined structural and compositional properties.

Poly-lactic acid nanoparticles (PLA-NP) promote physiological modifications in lung epithelial cells and are internalized by clathrin-coated pits and lipid rafts.

BACKGROUND: Poly-lactic acid nanoparticles (PLA-NP) are a type of polymeric NP, frequently used as nanomedicines, which have advantages over metallic NP such as the ability to maintain therapeutic drug levels for sustained periods of time. Despite PLA-NP being considered biocompatible, data concerning alterations in cellular physiology are scarce. METHODS: We conducted an extensive evaluation of PLA-NP biocompatibility in human lung epithelial A549 cells using high throughput screening and more complex methodologies. These included measurements of cytotoxicity, cell viability, immunomodulatory potential, and effects upon the cells' proteome. We used non- and green-fluorescent PLA-NP with 63 and 66 nm diameters, respectively. Cells were exposed with concentrations of 2, 20, 100 and 200 µg/mL, for 24, 48 and 72 h, in most experiments. Moreover, possible endocytic mechanisms of internalization of PLA-NP were investigated, such as those involving caveolae, lipid rafts, macropinocytosis and clathrin-coated pits. RESULTS: Cell viability and proliferation were not altered in response to PLA-NP. Multiplex analysis of secreted mediators revealed a low-level reduction of IL-12p70 and vascular epidermal growth factor (VEGF) in response to PLA-NP, while all other mediators assessed were unaffected. However, changes to the cells' proteome were observed in response to PLA-NP, and, additionally, the cellular stress marker miR155 was found to reduce. In dual exposures of staurosporine (STS) with PLA-NP, PLA-NP enhanced susceptibility to STS-induced cell death. Finally, PLA-NP were rapidly internalized in association with clathrin-coated pits, and, to a lesser extent, with lipid rafts. CONCLUSIONS: These data demonstrate that PLA-NP are internalized and, in general, tolerated by A549 cells, with no cytotoxicity and no secretion of pro-inflammatory mediators. However, PLA-NP exposure may induce modification of biological functions of A549 cells, which should be considered when designing drug delivery systems. Moreover, the pathways of PLA-NP internalization we detected could contribute to the improvement of selective uptake strategies.

Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.

The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-ß and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-ß and TNF-α measurements.