A roadmap towards safe and sustainable by design nanotechnology: Implementation for nano-silver-based antimicrobial textile coatings production by ASINA project.

This report demonstrates a case study within the ASINA project, aimed at instantiating a roadmap with quantitative metrics for Safe(r) and (more) Sustainable by Design (SSbD) options. We begin with a description of ASINA's methodology across the product lifecycle, outlining the quantitative elements within: Physical-Chemical Features (PCFs), Key Decision Factors (KDFs), and Key Performance Indicators (KPIs). Subsequently, we delve in a proposed decision support tool for implementing the SSbD objectives across various dimensions-functionality, cost, environment, and human health safety-within a broader European context. We then provide an overview of the technical processes involved, including design rationales, experimental procedures, and tools/models developed within ASINA in delivering nano-silver-based antimicrobial textile coatings. The result is pragmatic, actionable metrics intended to be estimated and assessed in future SSbD applications and to be adopted in a common SSbD roadmap aligned with the EU's Green Deal objectives. The methodological approach is transparently and thoroughly described to inform similar projects through the integration of KPIs into SSbD and foster data-driven decision-making. Specific results and project data are beyond this work's scope, which is to demonstrate the ASINA roadmap and thus foster SSbD-oriented innovation in nanotechnology.

A comparison of dermal toxicity models; assessing suitability for safe(r)-by-design decision-making and for screening nanomaterial hazards.

The objective of Safe-by-Design (SbD) is to support the development of safer products and production processes, and enable safe use throughout a materials' life cycle; an intervention at an early stage of innovation can greatly benefit industry by reducing costs associated with the development of products later found to elicit harmful effects. Early hazard screening can support this process, and is needed for all of the expected nanomaterial exposure routes, including inhalation, ingestion and dermal. In this study, we compare in vitro and ex vivo cell models that represent dermal exposures (including HaCaT cells, primary keratinocytes, and reconstructed human epidermis (RhE)), and when possible consider these in the context of regulatory accepted OECD TG for in vitro dermal irritation. Various benchmark nanomaterials were used to assess markers of cell stress in each cell model. In addition, we evaluated different dosing strategies that have been used when applying the OECD TG for dermal irritation in assessment of nanomaterials, and how inconsistencies in the approach used can have considerable impact of the conclusions made. Although we could not demonstrate alignment of all models used, there was an indication that the simpler in vitro cell model aligned more closely with RhE tissue than ex vivo primary keratinocytes, supporting the use of HaCaT cells for screening of dermal toxicity of nanomaterials and in early-stage SbD decision-making.

Perinatal foodborne titanium dioxide exposure-mediated dysbiosis predisposes mice to develop colitis through life.

BACKGROUND: Perinatal exposure to titanium dioxide (TiO2), as a foodborne particle, may influence the intestinal barrier function and the susceptibility to develop inflammatory bowel disease (IBD) later in life. Here, we investigate the impact of perinatal foodborne TiO2 exposure on the intestinal mucosal function and the susceptibility to develop IBD-associated colitis. Pregnant and lactating mother mice were exposed to TiO2 until pups weaning and the gut microbiota and intestinal barrier function of their offspring was assessed at day 30 post-birth (weaning) and at adult age (50 days). Epigenetic marks was studied by DNA methylation profile measuring the level of 5-methyl-2'-deoxycytosine (5-Me-dC) in DNA from colic epithelial cells. The susceptibility to develop IBD has been monitored using dextran-sulfate sodium (DSS)-induced colitis model. Germ-free mice were used to define whether microbial transfer influence the mucosal homeostasis and subsequent exacerbation of DSS-induced colitis. RESULTS: In pregnant and lactating mice, foodborne TiO2 was able to translocate across the host barriers including gut, placenta and mammary gland to reach embryos and pups, respectively. This passage modified the chemical element composition of foetus, and spleen and liver of mothers and their offspring. We showed that perinatal exposure to TiO2 early in life alters the gut microbiota composition, increases the intestinal epithelial permeability and enhances the colonic cytokines and myosin light chain kinase expression. Moreover, perinatal exposure to TiO2 also modifies the abilities of intestinal stem cells to survive, grow and generate a functional epithelium. Maternal TiO2 exposure increases the susceptibility of offspring mice to develop severe DSS-induced colitis later in life. Finally, transfer of TiO2-induced microbiota dysbiosis to pregnant germ-free mice affects the homeostasis of the intestinal mucosal barrier early in life and confers an increased susceptibility to develop colitis in adult offspring. CONCLUSIONS: Our findings indicate that foodborne TiO2 consumption during the perinatal period has negative long-lasting consequences on the development of the intestinal mucosal barrier toward higher colitis susceptibility. This demonstrates to which extent environmental factors influence the microbial-host interplay and impact the long-term mucosal homeostasis.

LipoParticles: a lipid membrane coating onto polymer particles to enhance the internalization in osteoblast cells.

LipoParticles, core-shell assemblies consisting of a polymer core coated by a lipid membrane, are promising carriers for drug delivery applications with intracellular targets. This is of great interest since it is actually challenging to treat infections involving intracellular bacteria such as bone and joint infections where the bacteria are hidden in osteoblast cells. The present work reports for the first time to the best of our knowledge the proof of enhanced internalization of particles in osteoblast cells thanks to a lipid coating of particles (= LipoParticles). The ca. 300 nm-sized assemblies were elaborated by reorganization of liposomes (composed of DPPC/DPTAP 10/90 mol/mol) onto the surface of poly(lactic-co-glycolic acid) (PLGA) particles, and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and zetametry. Optimization of these assemblies was also performed by adding poly(ethylene glycol) (PEG) chains on their surface (corresponding to a final formulation of DPPC/DPTAP/DPPE-PEG5000 8/90/2 mol/mol/mol). Interestingly, this provided them colloidal stability after their 20-fold dilution in PBS or cell culture medium, and made possible their freeze-drying without forming aggregates after their re-hydration. Their non-cytotoxicity towards a human osteoblast cell line (MG63) was also demonstrated. The enhanced internalization of LipoParticles in this MG63 cell line, in comparison with PLGA particles, was proven by observations with a confocal laser scanning microscope, as well as by flow cytometry assays. Finally, this efficient internalization of LipoParticles in MG63 cells was confirmed by TEM on ultrathin sections, which also revealed localization close to intracellular Staphylococcus aureus.

Nanomaterial genotoxicity evaluation using the high-throughput p53-binding protein 1 (53BP1) assay.

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies.

Hazard, Distribution and Exposure of Particulate Pollution from Indoor and Outdoor Environments.

Air is an essential natural resource for life [...].

Data-Driven Quantitative Intrinsic Hazard Criteria for Nanoproduct Development in a Safe-by-Design Paradigm: A Case Study of Silver Nanoforms.

The current European (EU) policies, that is, the Green Deal, envisage safe and sustainable practices for chemicals, which include nanoforms (NFs), at the earliest stages of innovation. A theoretically safe and sustainable by design (SSbD) framework has been established from EU collaborative efforts toward the definition of quantitative criteria in each SSbD dimension, namely, the human and environmental safety dimension and the environmental, social, and economic sustainability dimensions. In this study, we target the safety dimension, and we demonstrate the journey toward quantitative intrinsic hazard criteria derived from findable, accessible, interoperable, and reusable data. Data were curated and merged for the development of new approach methodologies, that is, quantitative structure-activity relationship models based on regression and classification machine learning algorithms, with the intent to predict a hazard class. The models utilize system (i.e., hydrodynamic size and polydispersity index) and non-system (i.e., elemental composition and core size)-dependent nanoscale features in combination with biological in vitro attributes and experimental conditions for various silver NFs, functional antimicrobial textiles, and cosmetics applications. In a second step, interpretable rules (criteria) followed by a certainty factor were obtained by exploiting a Bayesian network structure crafted by expert reasoning. The probabilistic model shows a predictive capability of ≈78% (average accuracy across all hazard classes). In this work, we show how we shifted from the conceptualization of the SSbD framework toward the realistic implementation with pragmatic instances. This study reveals (i) quantitative intrinsic hazard criteria to be considered in the safety aspects during synthesis stage, (ii) the challenges within, and (iii) the future directions for the generation and distillation of such criteria that can feed SSbD paradigms. Specifically, the criteria can guide material engineers to synthesize NFs that are inherently safer from alternative nanoformulations, at the earliest stages of innovation, while the models enable a fast and cost-efficient in silico toxicological screening of previously synthesized and hypothetical scenarios of yet-to-be synthesized NFs.

Physicochemical Transformations of Silver Nanoparticles in the Oro-Gastrointestinal Tract Mildly Affect Their Toxicity to Intestinal Cells In Vitro: An AOP-Oriented Testing Approach.

The widespread use of silver nanoparticles (Ag NPs) in food and consumer products suggests the relevance of human oral exposure to these nanomaterials (NMs) and raises the possibility of adverse effects in the gastrointestinal tract. The aim of this study was to investigate the toxicity of Ag NPs in a human intestinal cell line, either uncoated or coated with polyvinylpyrrolidone (Ag PVP) or hydroxyethylcellulose (Ag HEC) and digested in simulated gastrointestinal fluids. Physicochemical transformations of Ag NPs during the different stages of in vitro digestion were identified prior to toxicity assessment. The strategy for evaluating toxicity was constructed on the basis of adverse outcome pathways (AOPs) showing Ag NPs as stressors. It consisted of assessing Ag NP cytotoxicity, oxidative stress, genotoxicity, perturbation of the cell cycle and apoptosis. Ag NPs caused a concentration-dependent loss of cell viability and increased the intracellular level of reactive oxygen species as well as DNA damage and perturbation of the cell cycle. In vitro digestion of Ag NPs did not significantly modulate their toxicological impact, except for their genotoxicity. Taken together, these results indicate the potential toxicity of ingested Ag NPs, which varied depending on their coating but did not differ from that of non-digested NPs.

Preliminary Toxicological Analysis in a Safe-by-Design and Adverse Outcome Pathway-Driven Approach on Different Silver Nanoparticles: Assessment of Acute Responses in A549 Cells.

Silver nanoparticles (Ag NPs) are among the most widely used metal-based nanomaterials (NMs) and their applications in different products, also as antibacterial additives, are increasing. In the present manuscript, according to an adverse outcome pathway (AOP) approach, we tested two safe-by-design (SbD) newly developed Ag NPs coated with hydroxyethyl cellulose (HEC), namely AgHEC powder and AgHEC solution. These novel Ag NPs were compared to two reference Ag NPs (naked and coated with polyvinylpyrrolidone-PVP). Cell viability, inflammatory response, reactive oxygen species, oxidative DNA damage, cell cycle, and cell-particle interactions were analyzed in the alveolar in vitro model, A549 cells. The results show a different toxicity pattern of the novel Ag NPs compared to reference NPs and that between the two novel NPs, the AgHEC solution is the one with the lower toxicity and to be further developed within the SbD framework.

The State of the Art and Challenges of In Vitro Methods for Human Hazard Assessment of Nanomaterials in the Context of Safe-by-Design.

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing.

Putative adverse outcome pathways for silver nanoparticle toxicity on mammalian male reproductive system: a literature review.

BACKGROUND: Adverse outcome pathways (AOPs) are conceptual frameworks that organize knowledge about biological interactions and toxicity mechanisms. They present a sequence of events commencing with initial interaction(s) of a stressor, which defines the perturbation in a biological system (molecular initiating event, MIE), and a dependent series of key events (KEs), ending with an adverse outcome (AO). AOPs have recently become the subject of intense studies in a view to better understand the mechanisms of nanomaterial (NM) toxicity. Silver nanoparticles (Ag NPs) are one of the most explored nanostructures and are extensively used in various application. This, in turn, has increased the potential for interactions of Ag NPs with environments, and toxicity to human health. The aim of this study was to construct a putative AOPs (pAOP) related to reproductive toxicity of Ag NPs, in order to lay the groundwork for a better comprehension of mechanisms affecting both undesired toxicity (against human cell) and expected toxicity (against microorganisms). METHODS: PubMed and Scopus were systematically searched for peer-reviewed studies examining reproductive toxicity potential of Ag NPs. The quality of selected studies was assessed through ToxRTool. Eventually, forty-eight studies published between 2005 and 2022 were selected to identify the mechanisms of Ag NPs impact on reproductive function in human male. The biological endpoints, measurements, and results were extracted from these studies. Where possible, endpoints were assigned to a potential KE and an AO using expert judgment. Then, KEs were classified at each major level of biological organization. RESULTS: We identified the impairment of intracellular SH-containing biomolecules, which are major cellular antioxidants, as a putative MIE, with subsequent KEs defined as ROS accumulation, mitochondrial damage, DNA damage and lipid peroxidation, apoptosis, reduced production of reproductive hormones and reduced quality of sperm. These successive KEs may result in impaired male fertility (AO). CONCLUSION: This research recapitulates and schematically represents complex literature data gathered from different biological levels and propose a pAOP related to the reproductive toxicity induced by AgNPs. The development of AOPs specific to NMs should be encouraged in order to provide new insights to gain a better understanding of NP toxicity.

Nanostructured electrodes based on multiwalled carbon nanotube/glyconanoparticles for the specific immobilization of bilirubin oxidase: Application to the electrocatalytic O2 reduction.

Here we describe the design and the characterization of novel electrode materials consisting of multi-walled carbon nanotubes coated with glyconanoparticles (GNPs) functionalized with anthraquinone sulfonate. The resulting modified electrodes were characterized by scanning electron microscopy and cyclic voltammetry. Their electrochemical behavior reveals a stable pH-dependent redox signal characteristic of anthraquinone sulfonate. Immobilization of bilirubin oxidase on these three-dimensional electrodes leads to the electroenzymatic reduction of O2 to water with an onset potential of 0.5 V/SCE (saturated calomel electrode). A catalytic cathodic current of 174 µA (0.88 mA cm-2) at 0.1 V/SCE, demonstrates that glyconanoparticles modified by anthraquinone sulfonate were able to interact and orientate bilirubin oxidase by electrostatic interactions.

Selective plane illumination microscope dedicated to volumetric imaging in microfluidic chambers.

In this article, we are presenting an original selective plane illumination fluorescence microscope dedicated to image "Organ-on-chip"-like biostructures in microfluidic chips. In order to be able to morphologically analyze volumetric samples in development at the cellular scale inside microfluidic chambers, the setup presents a compromise between relatively large field of view (∼ 200 µm) and moderate resolution (∼ 5 µm). The microscope is based on a simple design, built around the chip and its microfluidic environment to allow 3D imaging inside the chip. In particular, the sample remains horizontally avoiding to disturb the fluidics phenomena. The experimental setup, its optical characterization and the first volumetric images are reported.

Physico-Chemical Transformation and Toxicity of Multi-Shell InP Quantum Dots under Simulated Sunlight Irradiation, in an Environmentally Realistic Scenario.

Quantum dots (QDs) are widely used in optoelectronics, lighting, and photovoltaics leading to their potential release into the environment. The most promising alternative to the highly toxic cadmium selenide (CdSe) QDs are indium phosphide (InP) QDs, which show reduced toxicity and comparable optical and electronic properties. QD degradation leads to the release of toxic metal ions into the environment. Coating the QD core with robust shell(s) composed of another semi-conductor material enhances their properties and protects the QD from degradation. We recently developed double-shelled InP QDs, which proved to be less toxic than single-shell QDs. In the present study, we confirm their reduced cytotoxicity, with an LC50 at 77 nM for pristine gradient shell QDs and >100 nM for pristine thin and thick shell QDs. We also confirm that these three QDs, when exposed to simulated sunlight, show greater cytotoxicity compared to pristine ones, with LC50 ranging from 15 to 23 nM. Using a combination of spectroscopic and microscopic techniques, we characterize the degradation kinetics and transformation products of single- and double-shell QDs, when exposed to solar light at high temperature, simulating environmental conditions. Non-toxic pristine QDs degrade to form toxic In−phosphate, In−carboxylate, Zn−phosphate, and oxidized Se, all of which precipitate as heterogeneous deposits. Comparison of their degradation kinetics highlights that the QDs bearing the thickest ZnS outer shell are, as expected, the most resistant to photodegradation among the three tested QDs, as gradient shell, thin shell, and thick shell QDs lose their optical properties in less than 15 min, 60 min, and more than 90 min, respectively. They exhibit the highest photoluminescence efficiency, i.e., the best functionality, with a photoluminescence quantum yield in aqueous solution of 24%, as compared to 18% for the gradient shell and thin shell QDs. Therefore, they can be considered as safer-by-design QDs.

Pre-validation of a reporter gene assay for oxidative stress for the rapid screening of nanobiomaterials.

Engineered nanomaterials have been found to induce oxidative stress. Cellular oxidative stress, in turn, can result in the induction of antioxidant and detoxification enzymes which are controlled by the nuclear erythroid 2-related factor 2 (NRF2) transcription factor. Here, we present the results of a pre-validation study which was conducted within the frame of BIORIMA ("biomaterial risk management") an EU-funded research and innovation project. For this we used an NRF2 specific chemically activated luciferase expression reporter gene assay derived from the human U2OS osteosarcoma cell line to screen for the induction of the NRF2 mediated gene expression following exposure to biomedically relevant nanobiomaterials. Specifically, we investigated Fe3O4-PEG-PLGA nanomaterials while Ag and TiO2 "benchmark" nanomaterials from the Joint Research Center were used as reference materials. The viability of the cells was determined by using the Alamar blue assay. We performed an interlaboratory study involving seven different laboratories to assess the applicability of the NRF2 reporter gene assay for the screening of nanobiomaterials. The latter work was preceded by online tutorials to ensure that the procedures were harmonized across the different participating laboratories. Fe3O4-PEG-PLGA nanomaterials were found to induce very limited NRF2 mediated gene expression, whereas exposure to Ag nanomaterials induced NRF2 mediated gene expression. TiO2 nanomaterials did not induce NRF2 mediated gene expression. The variability in the results obtained by the participating laboratories was small with mean intra-laboratory standard deviation of 0.16 and mean inter laboratory standard deviation of 0.28 across all NRF2 reporter gene assay results. We conclude that the NRF2 reporter gene assay is a suitable assay for the screening of nanobiomaterial-induced oxidative stress responses.

Magnetic particles for triggering insulin release in INS-1E cells subjected to a rotating magnetic field.

Diabetes is a major global health threat. Both academics and industry are striving to develop effective treatments for this disease. In this work, we present a new approach to induce insulin release from ß-islet pancreatic cells (INS-1E) by mechanical stimulation. Two types of experiments were carried out. First, a local stimulation was performed by dispersing anisotropic magnetic particles within the cell medium, which settled down almost immediately on cell plasma membranes. Application of a low frequency magnetic field (up to 40 Hz) generated by a custom-made magnetic device resulted in oscillations of these particles, which then exerted a mechanical constraint on the cell plasma membranes. The second type of experiment consisted of a global stimulation, where cells were grown on magneto-elastic membranes composed of a biocompatible polymer with embedded magnetic particles. Upon application of a rotating magnetic field, magnetic particles within the membrane were attracted towards the field source, resulting in the membrane's vibrations being transmitted to the cells grown on it. In both experiments, the cell response to these mechanical stimulations caused by application of the variable magnetic field was quantified via the measurement of insulin release in the growth medium. We demonstrated that the mechanical action induced by the motion of magnetic particles or by membrane vibrations was an efficient stimulus for insulin granule secretion from ß-cells. This opens a wide range of possible applications including the design of a system which triggers insulin secretion by ß-islet pancreatic cells on demand.

Immediate and Sustained Effects of Cobalt and Zinc-Containing Pigments on Macrophages.

Pigments are among the oldest nanoparticulate products known to mankind, and their use in tattoos is also very old. Nowadays, 25% of American people aged 18 to 50 are tattooed, which poses the question of the delayed effects of tattoos. In this article, we investigated three cobalt [Pigment Violet 14 (purple color)] or cobalt alloy pigments [Pigment Blue 28 (blue color), Pigment Green 14 (green color)], and one zinc pigment [Pigment White 4 (white color)] which constitute a wide range of colors found in tattoos. These pigments contain microparticles and a significant proportion of submicroparticles or nanoparticles (in either aggregate or free form). Because of the key role of macrophages in the scavenging of particulate materials, we tested the effects of cobalt- and zinc-based pigments on the J774A.1 macrophage cell line. In order to detect delayed effects, we compared two exposure schemes: acute exposure for 24 hours and an exposure for 24 hours followed by a 3-day post-exposure recovery period. The conjunction of these two schemes allowed for the investigation of the delayed or sustained effects of pigments. All pigments induced functional effects on macrophages, most of which were pigment-dependent. For example, Pigment Green 19, Pigment Blue 28, and Pigment White 4 showed a delayed alteration of the phagocytic capacity of cells. Moreover, all the pigments tested induced a slight but significant increase in tumor necrosis factor secretion. This effect, however, was transitory. Conversely, only Pigment Blue 28 induced both a short and sustained increase in interleukin 6 secretion. Results showed that in response to bacterial stimuli (LPS), the secretion of tumor necrosis factor and interleukin 6 declined after exposure to pigments followed by a recovery period. For chemoattractant cytokines (MCP-1 or MIP-1α), delayed effects were observed with a secretion decreased in presence of Pigment Blue 28 and Pigment violet 14, both with or without LPS stimuli. The pigments also induced persisting changes in some important macrophage membrane markers such as CD11b, an integrin contributing to cell adhesion and immunological tolerance. In conclusion, the pigments induced functional disorders in macrophages, which, in some cases, persist long after exposure, even at non-toxic doses.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

The laccase mediator system at carbon nanotubes for anthracene oxidation and femtomolar electrochemical biosensing.

We investigated the use of POXA1b laccase from Pleurotus ostreatus for the oxidation of anthracene into anthraquinone. We show that different pathways can occur depending on the nature of the redox mediator combined to laccase, leading to different structural isomers. The laccase combined with 2,2'-azine-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) leads to the formation of 1,4-anthraquinone and/or 1,2-anthraquinone. The unprecedented role of carbon nanotubes (CNTs) as redox mediators for oxidation of anthracene into 9,10-anthraquinone is shown and corroborated by density-functional theory (DFT) calculations. Owing to the efficient adsorption of anthraquinones at CNT electrodes, anthracene can be detected with low limit-of-detection using either laccase in solution, CNT-supported laccase or laccase immobilized at magnetic beads exploiting the adhesive property of a chimeric hydrophobin-laccase.