Boron Nitride Nanosheets Induce Lipid Accumulation and Autophagy in Human Alveolar Lung Epithelial Cells Cultivated at Air-Liquid Interface.

Hexagonal boron nitride (hBN) is an emerging 2D material attracting significant attention due to its superior electrical, chemical, and therapeutic properties. However, inhalation toxicity mechanisms of hBN in human lung cells are poorly understood. Here, cellular interaction and effects of hBN nanosheets is investigated in alveolar epithelial cells cultured on porous inserts and exposed under air-liquid interface conditions for 24 h. hBN is taken up by the cells as determined in a label-free manner via RAMAN-confocal microscopy, ICP-MS, TEM, and SEM-EDX. No significant (p > 0.05) effects are observed on cell membrane integrity (LDH release), epithelial barrier integrity (TEER), interleukin-8 cytokine production or reactive oxygen production at tested dose ranges (1, 5, and 10 µg cm-2). However, it is observed that an enhanced accumulation of lipid granules in cells indicating the effect of hBN on lipid metabolism. In addition, it is observed that a significant (p < 0.05) and dose-dependent (5 and 10 µg cm-2) induction of autophagy in cells after exposure to hBN, potentially associated with the downstream processing and breakdown of excess lipid granules to maintain lipid homeostasis. Indeed, lysosomal co-localization of lipid granules supporting this argument is observed. Overall, the results suggest that the continuous presence of excess intracellular lipids may provoke adverse outcomes in the lungs.

Approach toward In Vitro-Based Human Toxicity Effect Factors for the Life Cycle Impact Assessment of Inhaled Low-Solubility Particles.

Today's scarcity of animal toxicological data for nanomaterials could be lifted by substituting in vivo data with in vitro data to calculate nanomaterials' effect factors (EF) for Life Cycle Assessment (LCA). Here, we present a step-by-step procedure to calculate in vitro-to-in vivo extrapolation factors to estimate human Benchmark Doses and subsequently in vitro-based EFs for several inhaled nonsoluble nanomaterials. Based on mouse data, the in vitro-based EF of TiO2 is between 2.76 · 10-4 and 1.10 · 10-3 cases/(m2/g·kg intake), depending on the aerodynamic size of the particle, which is in good agreement with in vivo-based EFs (1.51 · 10-4-5.6 · 10-2 cases/(m2/g·kg intake)). The EF for amorphous silica is in a similar range as for TiO2, but the result is less robust due to only few in vivo data available. The results based on rat data are very different, confirming the importance of selecting animal species representative of human responses. The discrepancy between in vivo and in vitro animal data in terms of availability and quality limits the coverage of further nanomaterials. Systematic testing on human and animal cells is needed to reduce the variability in toxicological response determined by the differences in experimental conditions, thus helping improve the predictivity of in vitro-to-in vivo extrapolation factors.

Tailoring Design of Nanomaterials and Systems to Individualize Patient Treatments.

Nanomedicine encompasses usage of materials smaller than 100 nm for diagnosis, monitoring and treatment of disease. A frequent application of these materials is in reformulation of active drugs, which were previously approved for clinical use. As illustrated with chemotherapeutics, delivery of a drug within a nanocarrier can represent a clear clinical benefit as it can increase its targeted uptake and reduce the off-target toxicity. Matching nanomedicine treatments with patient-specific biomarkers provides an exciting prospect for moving the filed towards precision medicine. In parallel, a strong potential for personalized treatments comes from employing nanomaterials for the delivery of patient-tailored biologically active molecules. Recent research and clinical data have highlighted mRNA and siRNA molecules, as well as short peptides, as powerful new drug classes that can be designed according to patient profiles and effectively delivered within nanoparticles. Particles used for therapeutic delivery are based on biodegradable and safe materials, frequently lipids and polymers, which can be further functionalized into more complex forms. Currently, there is a strong interest in developing specific nanocarrier formulations which can achieve optimal delivery of active molecules to targeted cells while reducing unwanted side-effects. Here, we discuss recent developments and future perspectives in the nanomedicine field and specifically highlight innovative approaches for the personalized patient treatments.

Community Masks - from an Emergency Solution to an Innovation Booster for the Textile Industry.

The COVID-19 pandemic resulted in shortages of personal protective equipment and medical devices in the initial phase. Agile small and medium-sized enterprises from regional textile industries reacted quickly. They delivered alternative products such as textile-based community masks in collaboration with industrial partners and research institutes from various sectors. The current mask materials and designs were further improved by integrating textiles with antiviral and antimicrobial properties and enhanced protection and comfort by novel textile/membrane combinations, key factors to increase the acceptance and compliance of mask wearing. The innocuity and sustainability of masks, as well as taking into account particular needs of vulnerable persons in our society, are new fields for textile-based innovations. These innovations developed for the next generation of facemasks have a high adaptability to other product segments, which make textiles an attractive material for hygienic applications and beyond.

Investigating the effects of differently produced synthetic amorphous silica (E 551) on the integrity and functionality of the human intestinal barrier using an advanced in vitro co-culture model.

E 551, also known as synthetic amorphous silica (SAS), is the second most produced food additive. However, according to the re-evaluation of E 551 by the European Food Safety Authority (EFSA) in 2018, the amount of available data on the oral toxicity of food grade E 551 is still insufficient for reliable risk assessment. To close this gap, this study aimed to investigate six food-grade SAS with distinct physicochemical properties on their interaction with the intestinal barrier using advanced in vitro intestinal co-cultures and to identify potential structure-activity relationships. A mucus-secreting Caco-2/HT-29/Raji co-culture model was treated with up to 50 µg/ml SAS for 48 h, which represents a dose range relevant to dietary exposure. No effects on cell viability, barrier integrity, microvilli function or the release of inflammatory cytokine were detected after acute exposure. Slight biological responses were observed for few SAS materials on iron uptake and gene expression levels of mucin 1 and G-protein coupled receptor 120 (GPR120). There was no clear correlation between SAS properties (single or combined) and the observed biological responses. Overall, this study provides novel insights into the short-term impact of food-relevant SAS with distinct characteristics on the intestinal epithelium including a range of intestine-specific functional endpoints. In addition, it highlights the importance of using advanced intestinal co-cultures embracing relevant cell types as well as a protective mucus barrier to achieve a comprehensive understanding of the biological response of food additives at the intestinal barrier in vitro.

Understanding Nanomaterial Biotransformation: An Unmet Challenge to Achieving Predictive Nanotoxicology.

More than a decade has passed since the first concepts of predictive nanotoxicology were formulated. During this time, many advancements have been achieved in multiple disciplines, including the success stories of the fiber paradigm and the oxidative stress paradigm. However, important knowledge gaps are slowing down the development of predictive nanotoxicology and require a mutidisciplinary effort to be overcome. Among these gaps, understanding, reproducing, and modeling of nanomaterial biotransformation in biological environments is a central challenge, both in vitro and in silico. This dynamic and complex process is still a challenge for today's bioanalytics. This work explores and discusses selected approaches of the multidisciplinary efforts taken in the last decade and the challenges that remain unmet, in particular concerning nanomaterial biotransformation. It highlights some future advancements that, together, can help to understand such complex processes and accelerate the development of predictive nanotoxicology.

Exploring Flow Cytometry-Based Micronucleus Scoring for Reliable Nanomaterial Genotoxicity Assessment.

The increased use of engineered nanomaterials (ENM) such as SiO2 and TiO2 in industrial products, especially in food, raises concerns with regard to their effect on human health. In particular, ENM-induced genotoxicity is crucial to investigate, since DNA damage can cause induction or promotion of carcinogenesis. However, current in vitro and in vivo nanogenotoxicological data are highly contradictory, which impedes interpretation and extrapolation. Hence, robust, reliable, and ideally scalable in vitro methods for nanogenotoxicity assessment are of great interest. This work aimed at evaluating the suitability of flow cytometry-based micronuclei scoring for reliable nanogenotoxicological assessment in human intestinal cells. Therefore, we have evaluated the genotoxicity of differently sized SiO2 and TiO2 from different sources (food-relevant, commercially available, and laboratory-synthesized) using the well-established alkaline single cell gel electrophoresis (Comet assay) and the micronucleus (MN) assay employing a flow cytometric readout. Our study demonstrates that physiologically relevant doses of several types of SiO2 and TiO2 did not cause genotoxicity, as assessed by the Comet assay, and the MN flow cytometry assay under the particular experimental conditions described. To improve data reliability, we identified ENM-induced interferences with flow cytometric scoring employing a set of interference controls, which is generally applicable for any nanomaterial and any cell line. In conclusion, flow cytometry-based MN scoring appears to be a promising methodology in nanogenotoxicity testing since data acquisition and analysis are significantly faster, highly scalable in terms of throughput, and less operator-dependent compared to the traditional microscopic evaluation. In particular, ENM-induced false-positive or false-negative results, which have not been addressed sufficiently in the literature, can be detected easily, thus enhancing data reliability.

Innovative Techniques and Strategies for a Reliable High-Throughput Genotoxicity Assessment.

Damage to DNA is a central mechanism to the initiation of carcinogenesis. As a consequence, precise DNA damage detection is essential for an effective risk assessment of xenobiotics and constitutes a powerful tool for human biomonitoring and early stage cancer risk assessment. Here we highlight four innovative approaches for determining genotoxicity in a reliable and in the future high-throughput manner. In this context, we discuss and evaluate recent improvements to well-established methods and present promising new techniques.

Cause-and-Effect Analysis as a Tool To Improve the Reproducibility of Nanobioassays: Four Case Studies.

One of the challenges in using in vitro data to understand the potential risks of engineered nanomaterials (ENMs) is that results often differ or are even contradictory among studies. While it is recognized that numerous factors can influence results produced by nanobioassays, there has not yet been a consistently used conceptual framework to identify key sources of variability in these assays. In this paper, we use cause-and-effect analysis to systematically describe sources of variability in four key in vitro nanobioassays: the 2',7'-dichlorofluorescein assay, an enzyme-linked immunosorbent assay for measuring interleukin-8, a flow cytometry assay (Annexin V/propidium iodide), and the Comet assay. These assays measure end points that can occur in cells impacted by ENMs through oxidative stress, a principle mechanism for ENM toxicity. The results from this analysis identify control measurements to test for potential artifacts or biases that could occur during conduct of these assays with ENMs. Cause-and-effect analysis also reveals additional measurements that could be performed either in preliminary experiments or each time the assay is run to increase confidence in the assay results and their reproducibility within and among laboratories. The approach applied here with these four assays can be used to support the development of a broad range of nanobioassays.

Factors influencing safety and efficacy of intravenous iron-carbohydrate nanomedicines: From production to clinical practice.

Iron deficiency is an important subclinical disease affecting over one billion people worldwide. A growing body of clinical records supports the use of intravenous iron-carbohydrate complexes for patients where iron replenishment is necessary and oral iron supplements are either ineffective or cannot be tolerated by the gastrointestinal tract. A critical characteristic of iron-carbohydrate drugs is the complexity of their core-shell structure, which has led to differences in the efficacy and safety of various iron formulations. This review describes parameters influencing the safety and effectiveness of iron-carbohydrate complexes during production, storage, handling, and clinical application. We summarized the physicochemical and biological assessments of commercially available iron carbohydrate nanomedicines to provide an overview of publicly available data. Further, we reviewed studies that described how subtle differences in the manufacturing process of iron-carbohydrate complexes can impact on the physicochemical, biological, and clinical outcomes of original product versus their intended copies or so-called iron "similar" products.

Nano-analytical characterization of endogenous minerals in healthy placental tissue: mineral distribution, composition and ultrastructure.

Despite its crucial role, the placenta is the least understood human organ. Recent clinical studies indicate a direct association between placental calcification and maternal and offspring health. This study reveals distinct characteristics of minerals formed during gestational ageing using cutting-edge nano-analytical characterization and paves the way for investigations focused on the identification of potential markers for disease risks in a clinical setting based on atypical placental mineral fingerprints.

Developmental Toxicity of Nanomaterials: Need for a Better Understanding of Indirect Effects.

While placental translocation and direct toxicity to fetal tissue of traversed nanomaterials has been a key focus of developmental toxicity studies, the release of maternal and fetal mediators that indirectly interfere with fetal development and health later in life lacks systematic insights and deserves special attention.

Gold nanoparticle distribution in advanced in vitro and ex vivo human placental barrier models.

BACKGROUND: Gold nanoparticles (AuNPs) are promising candidates to design the next generation NP-based drug formulations specifically treating maternal, fetal or placental complications with reduced side effects. Profound knowledge on AuNP distribution and effects at the human placental barrier in dependence on the particle properties and surface modifications, however, is currently lacking. Moreover, the predictive value of human placental transfer models for NP translocation studies is not yet clearly understood, in particular with regards to differences between static and dynamic exposures. To understand if small (3-4 nm) AuNPs with different surface modifications (PEGylated versus carboxylated) are taken up and cross the human placental barrier, we performed translocation studies in a static human in vitro co-culture placenta model and the dynamic human ex vivo placental perfusion model. The samples were analysed using ICP-MS, laser ablation-ICP-MS and TEM analysis for sensitive, label-free detection of AuNPs. RESULTS: After 24 h of exposure, both AuNP types crossed the human placental barrier in vitro, although in low amounts. Even though cellular uptake was higher for carboxylated AuNPs, translocation was slightly increased for PEGylated AuNPs. After 6 h of perfusion, only PEGylated AuNPs were observed in the fetal circulation and tissue accumulation was similar for both AuNP types. While PEGylated AuNPs were highly stable in the biological media and provided consistent results among the two placenta models, carboxylated AuNPs agglomerated and adhered to the perfusion device, resulting in different cellular doses under static and dynamic exposure conditions. CONCLUSIONS: Gold nanoparticles cross the human placental barrier in limited amounts and accumulate in placental tissue, depending on their size- and/or surface modification. However, it is challenging to identify the contribution of individual characteristics since they often affect colloidal particle stability, resulting in different biological interaction in particular under static versus dynamic conditions. This study highlights that human ex vivo and in vitro placenta models can provide valuable mechanistic insights on NP uptake and translocation if accounting for NP stability and non-specific interactions with the test system.

Characterisation of particles in solution - a perspective on light scattering and comparative technologies.

We present here a perspective detailing the current state-of-the-art technologies for the characterisation of nanoparticles (NPs) in liquid suspension. We detail the technologies involved and assess their applications in the determination of NP size and concentration. We also investigate the parameters that can influence the results and put forward a cause and effect analysis of the principle factors influencing the measurement of NP size and concentration by NP tracking analysis and dynamic light scattering, to identify areas where uncertainties in the measurement can arise. Also included are technologies capable of characterising NPs in solution, whose measurements are not based on light scattering. It is hoped that the manuscript, with its detailed description of the methodologies involved, will assist scientists in selecting the appropriate technology for characterising their materials and enabling them to comply with regulatory agencies' demands for accurate and reliable NP size and concentration data.

MyD88-dependent pro-interleukin-1ß induction in dendritic cells exposed to food-grade synthetic amorphous silica.

BACKGROUND: Dendritic cells (DCs) are specialized first-line sensors of foreign materials invading the organism. These sentinel cells rely on pattern recognition receptors such as Nod-like or Toll-like receptors (TLRs) to launch immune reactions against pathogens, but also to mediate tolerance to self-antigens and, in the intestinal milieu, to nutrients and commensals. Since inappropriate DC activation contributes to inflammatory diseases and immunopathologies, a key question in the evaluation of orally ingested nanomaterials is whether their contact with DCs in the intestinal mucosa disrupts this delicate homeostatic balance between pathogen defense and tolerance. Here, we generated steady-state DCs by incubating hematopoietic progenitors with feline McDonough sarcoma-like tyrosine kinase 3 ligand (Flt3L) and used the resulting immature DCs to test potential biological responses against food-grade synthetic amorphous silica (SAS) representing a common nanomaterial generally thought to be safe. RESULTS: Interaction of immature and unprimed DCs with food-grade SAS particles and their internalization by endocytic uptake fails to elicit cytotoxicity and the release of interleukin (IL)-1α or tumor necrosis factor-α, which were identified as master regulators of acute inflammation in lung-related studies. However, the display of maturation markers on the cell surface shows that SAS particles activate completely immature DCs. Also, the endocytic uptake of SAS particles into these steady-state DCs leads to induction of the pro-IL-1ß precursor, subsequently cleaved by the inflammasome to secrete mature IL-1ß. In contrast, neither pro-IL-1ß induction nor mature IL-1ß secretion occurs upon internalization of TiO2 or FePO4 nanoparticles. The pro-IL-1ß induction is suppressed by pharmacologic inhibitors of endosomal TLR activation or by genetic ablation of MyD88, a downstream adapter of TLR pathways, indicating that endosomal pattern recognition is responsible for the observed cytokine response to food-grade SAS particles. CONCLUSIONS: Our results unexpectedly show that food-grade SAS particles are able to directly initiate the endosomal MyD88-dependent pathogen pattern recognition and signaling pathway in steady-state DCs. The ensuing activation of immature DCs with de novo induction of pro-IL-1ß implies that the currently massive use of SAS particles as food additive should be reconsidered.

Cytotoxic effects of nanosilver are highly dependent on the chloride concentration and the presence of organic compounds in the cell culture media.

BACKGROUND: Nanosilver shows great promise for use in industrial, consumer or medical products because of its antimicrobial properties. However, the underlying mechanisms of the effects of silver nanoparticles on human cells are still controversial. Therefore, in the present study the influence of the chloride concentration and different serum content of culture media on the cytotoxic effects of nanosilver was systematically evaluated. RESULTS: Our results show that nanosilver toxicity was strongly affected by the composition of the culture media. The chloride concentration, as well as the carbon content affected the silver agglomeration and the complex formation. But also the dissolution of nanosilver and the availability of free silver ions (Ag+) were severely affected by the compositions of the culture media. Cells, only exposed to silver particles in suspension and dissolved silver complexes, did not show any effects under all conditions. Nanosilver agglomerates and silver complexes were not very soluble. Thus, cells growing on the bottom of the culture dishes were exposed to sedimented nanosilver agglomerates and precipitated silver complexes. Locally, the concentration of silver on the cell surface was very high, much higher compared the silver concentration in the bulk solution. The cytotoxic effects of nanosilver are therefore a combination of precipitated silver complexes and organic silver compounds rather than free silver ions. CONCLUSIONS: Silver coatings are used in health care products due to their bacteriostatic or antibacterial properties. The assessment of the toxicity of a certain compound is mostly done using in vitro assays. Therefore, cytotoxicity studies of nanosilver using human cell cultures have to be undertaken under well controlled and understood cultivations conditions in order to improve the compatibility of different studies. Especially when eukaryotic versus prokaryotic systems are compared for the evaluation of the use of nanosilver as antibacterial coatings for implants in order to prevent bacterial colonization.

Uptake of label-free graphene oxide by Caco-2 cells is dependent on the cell differentiation status.

BACKGROUND: Understanding the interaction of graphene-related materials (GRM) with human cells is a key to the assessment of their potential risks for human health. There is a knowledge gap regarding the potential uptake of GRM by human intestinal cells after unintended ingestion. Therefore the aim of our study was to investigate the interaction of label-free graphene oxide (GO) with the intestinal cell line Caco-2 in vitro and to shed light on the influence of the cell phenotype given by the differentiation status on cellular uptake behaviour. RESULTS: Internalisation of two label-free GOs with different lateral size and thickness by undifferentiated and differentiated Caco-2 cells was analysed by scanning electron microscopy and transmission electron microscopy. Semi-quantification of cells associated with GRM was performed by flow cytometry. Undifferentiated Caco-2 cells showed significant amounts of cell-associated GRM, whereas differentiated Caco-2 cells exhibited low adhesion of GO sheets. Transmission electron microscopy analysis revealed internalisation of both applied GO (small and large) by undifferentiated Caco-2 cells. Even large GO sheets with lateral dimensions up to 10 µm, were found internalised by undifferentiated cells, presumably by macropinocytosis. In contrast, no GO uptake could be found for differentiated Caco-2 cells exhibiting an enterocyte-like morphology with apical brush border. CONCLUSIONS: Our results show that the internalisation of GO is highly dependent on the cell differentiation status of human intestinal cells. During differentiation Caco-2 cells undergo intense phenotypic changes which lead to a dramatic decrease in GRM internalisation. The results support the hypothesis that the cell surface topography of differentiated Caco-2 cells given by the brush border leads to low adhesion of GO sheets and sterical hindrance for material uptake. In addition, the mechanical properties of GRM, especially flexibility of the sheets, seem to be an important factor for internalisation of large GO sheets by epithelial cells. Our results highlight the importance of the choice of the in vitro model to enable better in vitro-in vivo translation.

Multiparameter toxicity assessment of novel DOPO-derived organophosphorus flame retardants.

Halogen-free organophosphorus flame retardants are considered as replacements for the phased-out class of polybrominated diphenyl ethers (PBDEs). However, toxicological information on new flame retardants is still limited. Based on their excellent flame retardation potential, we have selected three novel 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) derivatives and assessed their toxicological profile using a battery of in vitro test systems in order to provide toxicological information before their large-scale production and use. PBDE-99, applied as a reference compound, exhibited distinct neuro-selective cytotoxicity at concentrations ≥10 µM. 6-(2-((6-oxido-6H-dibenzo[c,e][1,2]oxaphosphinin-6-yl)amino)ethoxy)-6H-dibenzo[c,e][1,2]oxaphosphinine 6-oxide (ETA-DOPO) and 6,6'-(ethane-1,2-diylbis(oxy))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EG-DOPO) displayed adverse effects at concentrations >10 µM in test systems reflecting the properties of human central and peripheral nervous system neurons, as well as in a set of non-neuronal cell types. DOPO and its derivative 6,6'-(ethane-1,2-diylbis(azanediyl))bis(6H-dibenzo[c,e][1,2]oxaphosphinine-6-oxide) (EDA-DOPO) were neither neurotoxic, nor did they exhibit an influence on neural crest cell migration, or on the integrity of human skin equivalents. The two compounds furthermore displayed no inflammatory activation potential, nor did they affect algae growth or daphnia viability at concentrations ≤400 µM. Based on the superior flame retardation properties, biophysical features suited for use in polyurethane foams, and low cytotoxicity of EDA-DOPO, our results suggest that it is a candidate for the replacement of currently applied flame retardants.

Release of copper-amended particles from micronized copper-pressure-treated wood during mechanical abrasion.

BACKGROUND: We investigated the particles released due to abrasion of wood surfaces pressure-treated with micronized copper azole (MCA) wood preservative and we gathered preliminary data on its in vitro cytotoxicity for lung cells. The data were compared with particles released after abrasion of untreated, water (0% MCA)-pressure-treated, chromated copper (CC)-pressure-treated wood, and varnished wood. Size, morphology, and composition of the released particles were analyzed. RESULTS: Our results indicate that the abrasion of MCA-pressure-treated wood does not cause an additional release of nanoparticles from the unreacted copper (Cu) carbonate nanoparticles from of the MCA formulation. However, a small amount of released Cu was detected in the nanosized fraction of wood dust, which could penetrate the deep lungs. The acute cytotoxicity studies were performed on a human lung epithelial cell line and human macrophages derived from a monocytic cell line. These cell types are likely to encounter the released wood particles after inhalation. CONCLUSIONS: Our findings indicate that under the experimental conditions chosen, MCA does not pose a specific additional nano-risk, i.e. there is no additional release of nanoparticles and no specific nano-toxicity for lung epithelial cells and macrophages.

Use of Cause-and-Effect Analysis to Design a High-Quality Nanocytotoxicology Assay.

An important consideration in developing standards and regulations that govern the production and use of commercial nanoscale materials is the development of robust and reliable measurements to monitor the potential adverse biological effects of such products. These measurements typically require cell-based and other biological assays that provide an assessment of the risks associated with the nanomaterial of interest. In this perspective, we describe the use of cause-and-effect (C&E) analysis to design robust, high quality cell-based assays to test nanoparticle-related cytotoxicity. C&E analysis of an assay system identifies the sources of variability that influence the test result. These sources can then be used to design control experiments that aid in establishing the validity of a test result. We demonstrate the application of C&E analysis to the commonly used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay. This is the first time to our knowledge that C&E analysis has been used to characterize a cell-based toxicity assay. We propose the use of a 96-well plate layout which incorporates a range of control experiments to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance, and demonstrate the performance of the assay using the plate layout in a case study. While the plate layout was formulated specifically for the MTS assay, it is applicable to other cytotoxicity, ecotoxicity (i.e., bacteria toxicity), and nanotoxicity assays after assay-specific modifications.