Exploiting the ferroaddiction of pancreatic cancer cells using Fe-doped nanoparticles.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with poor survival rates. Here, we evaluated iron-doped hydroxyapatite (FeHA) as a potential nanomedicine-based approach to combat PDAC. FeHA, in combination with a sublethal dose of the glutathione peroxidase 4 (GPX4) inhibitor RSL3, was found to trigger ferroptosis in KRAS mutant PANC-1 cells, but not in BxPC3 cells, while sparing normal human cells (fibroblasts and peripheral blood mononuclear cells). These findings were recapitulated in 3D spheroids generated using PDAC cells harboring wild-type versus mutant KRAS. Moreover, ferroptosis induction by FeHA plus RSL3 was reversed by the knockdown of STEAP3, a metalloreductase responsible for converting Fe3+ to Fe2+. Taken together, our data show that FeHA is capable of triggering cancer cell death in a KRAS-selective, STEAP3-dependent manner in PDAC cells.

Cytotoxicity assessment of exfoliated MoS2 using primary human mast cells and the progenitor cell-derived mast cell line LAD2.

Molybdenum disulfide is an emerging 2D material with several potential applications in medicine. Therefore, it is crucial to ascertain its biocompatibility. Mast cells are immune cells that are found in many organs and tissues in contact with the extracellular environment, and can be cultured from progenitor cells present in the bone marrow. Given the long period required for differentiation and proliferation of primary mast cells, human mast cell lines have emerged as a tractable model for biological and toxicological studies. Here, we compare two types of industrial MoS2 using CD34+-derived primary human mast cells and the LAD2 cell line. Minimal effects were observed on early-stage activation endpoints such as ß-hexosaminidase release and expression of surface markers of mast cell activation. Transmission electron microscopy revealed limited uptake of the tested materials. Overall, MoS2 was found to be biocompatible, and the LAD2 cell line was validated as a useful in vitro model of mast cells.

Hazard assessment of hexagonal boron nitride and hexagonal boron nitride reinforced thermoplastic polyurethane composites using human skin and lung cells.

Hexagonal boron nitride (hBN) is an emerging two-dimensional material attracting considerable attention in the industrial sector given its innovative physicochemical properties. Potential risks are associated mainly with occupational exposure where inhalation and skin contact are the most relevant exposure routes for workers. Here we aimed at characterizing the effects induced by composites of thermoplastic polyurethane (TPU) and hBN, using immortalized HaCaT skin keratinocytes and BEAS-2B bronchial epithelial cells. The composite was abraded using a Taber® rotary abraser and abraded TPU and TPU-hBN were also subjected to photo-Fenton-mediated degradation mimicking potential weathering across the product life cycle. Cells were exposed to the materials for 24 h (acute exposure) or twice per week for 4 weeks (chronic exposure) and evaluated with respect to material internalization, cytotoxicity, and proinflammatory cytokine secretion. Additionally, comprehensive mass spectrometry-based proteomics and metabolomics (secretomics) analyses were performed. Overall, despite evidence of cellular uptake of the material, no significant cellular and/or protein expression profiles alterations were observed after acute or chronic exposure of HaCaT or BEAS-2B cells, identifying only few pro-inflammatory proteins. Similar results were obtained for the degraded materials. These results support the determination of hazard profiles associated with cutaneous and pulmonary hBN-reinforced polymer composites exposure.

Nanosafety: a Perspective on Nano-Bio Interactions.

Engineered nanomaterials offer numerous benefits to society ranging from environmental remediation to biomedical applications such as drug or vaccine delivery as well as clean and cost-effective energy production and storage, and the promise of a more sustainable way of life. However, as nanomaterials of increasing sophistication enter the market, close attention to potential adverse effects on human health and the environment is needed. Here a critical perspective on nanotoxicological research is provided; the authors argue that it is time to leverage the knowledge regarding the biological interactions of nanomaterials to achieve a more comprehensive understanding of the human health and environmental impacts of these materials. Moreover, it is posited that nanomaterials behave like biological entities and that they should be regulated as such.

Environmental and Health Impacts of Graphene and Other Two-Dimensional Materials: A Graphene Flagship Perspective.

Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013-2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.

Two-dimensional molybdenum disulfide nanosheets evoke nitric oxide-dependent antibacterial effects.

Nanomaterials are currently being explored as novel antimicrobial agents. In this study, we first investigated the ability of two-dimensional (2D) molybdenum disulfide (MoS2) nanosheets to trigger neutrophil extracellular traps (NETs) using neutrophil-differentiated HL-60 cells as well as primary human peripheral blood neutrophils. We then addressed whether the MoS2 nanosheets themselves function as antibacterial agents. We found that MoS2 and Na2MoO4 both triggered NETs, as evidenced by the quantification of neutrophil elastase (NE) activity and immunofluorescence staining of extracellular NE, as well as scanning electron microscopy. The release of NETs was found to be nitric oxide (NO)-dependent. We also found that the MoS2 nanosheets but not the soluble salt prompted acellular NO production in the presence of NaNO2. The acellular generation of NO, suggestive of nanozyme properties of the MoS2 nanosheets, was demonstrated by electron paramagnetic resonance analysis. Electrochemical analysis using cyclic voltammetry confirmed the redox transition of the MoS2 nanosheets. Finally, MoS2 nanosheets inhibited the growth of Escherichia coli in the presence of sodium nitrate. Taken together, MoS2 nanosheets triggered cellular effects as well as acellular antibacterial effects, and we provided evidence for nitrite reductase-like properties of MoS2.

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.

Don't sweat the small stuff: a conversation about nanosafety.

Bengt Fadeel and Phil Sayre discuss lessons learned with respect to the safety assessment of nanomaterials, and provide a perspective on current and future challenges.

Exploiting Mass Spectrometry to Unlock the Mechanism of Nanoparticle-Induced Inflammasome Activation.

Nanoparticles (NPs) elicit sterile inflammation, but the underlying signaling pathways are poorly understood. Here, we report that human monocytes are particularly vulnerable to amorphous silica NPs, as evidenced by single-cell-based analysis of peripheral blood mononuclear cells using cytometry by time-of-flight (CyToF), while silane modification of the NPs mitigated their toxicity. Using human THP-1 cells as a model, we observed cellular internalization of silica NPs by nanoscale secondary ion mass spectrometry (nanoSIMS) and this was confirmed by transmission electron microscopy. Lipid droplet accumulation was also noted in the exposed cells. Furthermore, time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed specific changes in plasma membrane lipids, including phosphatidylcholine (PC) in silica NP-exposed cells, and subsequent studies suggested that lysophosphatidylcholine (LPC) acts as a cell autonomous signal for inflammasome activation in the absence of priming with a microbial ligand. Moreover, we found that silica NPs elicited NLRP3 inflammasome activation in monocytes, whereas cell death transpired through a non-apoptotic, lipid peroxidation-dependent mechanism. Together, these data further our understanding of the mechanism of sterile inflammation.

Label-free detection of polystyrene nanoparticles in Daphnia magna using Raman confocal mapping.

Micro- and nanoplastic pollution has emerged as a global environmental problem. Moreover, plastic particles are of increasing concern for human health. However, the detection of so-called nanoplastics in relevant biological compartments remains a challenge. Here we show that Raman confocal spectroscopy-microscopy can be deployed for the non-invasive detection of amine-functionalized and carboxy-functionalized polystyrene (PS) nanoparticles (NPs) in Daphnia magna. The presence of PS NPs in the gastrointestinal (GI) tract of D. magna was confirmed by using transmission electron microscopy. Furthermore, we investigated the ability of NH2-PS NPs and COOH-PS NPs to disrupt the epithelial barrier of the GI tract using the human colon adenocarcinoma cell line HT-29. To this end, the cells were differentiated for 21 days and then exposed to PS NPs followed by cytotoxicity assessment and transepithelial electrical resistance measurements. A minor disruption of barrier integrity was noted for COOH-PS NPs, but not for the NH2-PS NPs, while no overt cytotoxicity was observed for both NPs. This study provides evidence of the feasibility of applying label-free approaches, i.e., confocal Raman mapping, to study PS NPs in a biological system.

High-Dose Exposure to Polymer-Coated Iron Oxide Nanoparticles Elicits Autophagy-Dependent Ferroptosis in Susceptible Cancer Cells.

Ferroptosis, a form of iron-dependent, lipid peroxidation-driven cell death, has been extensively investigated in recent years, and several studies have suggested that the ferroptosis-inducing properties of iron-containing nanomaterials could be harnessed for cancer treatment. Here we evaluated the potential cytotoxicity of iron oxide nanoparticles, with and without cobalt functionalization (Fe2O3 and Fe2O3@Co-PEG), using an established, ferroptosis-sensitive fibrosarcoma cell line (HT1080) and a normal fibroblast cell line (BJ). In addition, we evaluated poly (ethylene glycol) (PEG)-poly(lactic-co-glycolic acid) (PLGA)-coated iron oxide nanoparticles (Fe3O4-PEG-PLGA). Our results showed that all the nanoparticles tested were essentially non-cytotoxic at concentrations up to 100 µg/mL. However, when the cells were exposed to higher concentrations (200-400 µg/mL), cell death with features of ferroptosis was observed, and this was more pronounced for the Co-functionalized nanoparticles. Furthermore, evidence was provided that the cell death triggered by the nanoparticles was autophagy-dependent. Taken together, the exposure to high concentrations of polymer-coated iron oxide nanoparticles triggers ferroptosis in susceptible human cancer cells.

Die hard: cell death mechanisms and their implications in nanotoxicology.

Cell death is a fundamental biological process, and its fine-tuned regulation is required for life. However, the complexity of regulated cell death is often reduced to a matter of live-dead discrimination. Here, we provide a perspective on programmed or regulated cell death, focusing on apoptosis, pyroptosis, necroptosis, and ferroptosis (the latter three cell death modalities are examples of regulated necrosis). We also touch on other, recently described manifestations of (pathological) cell death including cuproptosis. Furthermore, we address how engineered nanomaterials impact on regulated cell death. We posit that an improved understanding of nanomaterial-induced perturbations of cell death may allow for a better prediction of the consequences of human exposure and could also yield novel approaches by which to mitigate these effects. Finally, we provide examples of the harnessing of nanomaterials to achieve cancer cell killing through the induction of regulated cell death.

Graphene oxide elicits microbiome-dependent type 2 immune responses via the aryl hydrocarbon receptor.

The gut microbiome produces metabolites that interact with the aryl hydrocarbon receptor (AhR), a key regulator of immune homoeostasis in the gut1,2. Here we show that oral exposure to graphene oxide (GO) modulates the composition of the gut microbiome in adult zebrafish, with significant differences in wild-type versus ahr2-deficient animals. Furthermore, GO was found to elicit AhR-dependent induction of cyp1a and homing of lck+ cells to the gut in germ-free zebrafish larvae when combined with the short-chain fatty acid butyrate. To obtain further insights into the immune responses to GO, we used single-cell RNA sequencing to profile cells from whole germ-free embryos as well as cells enriched for lck. These studies provided evidence for the existence of innate lymphoid cell (ILC)-like cells3 in germ-free zebrafish. Moreover, GO endowed with a 'corona' of microbial butyrate triggered the induction of ILC2-like cells with attributes of regulatory cells. Taken together, this study shows that a nanomaterial can influence the crosstalk between the microbiome and immune system in an AhR-dependent manner.

Silver nanoparticles with excellent biocompatibility block pseudotyped SARS-CoV-2 in the presence of lung surfactant.

Silver (Ag) is known to possess antimicrobial properties which is commonly attributed to soluble Ag ions. Here, we showed that Ag nanoparticles (NPs) potently inhibited SARS-CoV-2 infection using two different pseudovirus neutralization assays. We also evaluated a set of Ag nanoparticles of different sizes with varying surface properties, including polyvinylpyrrolidone (PVP)-coated and poly (ethylene glycol) (PEG)-modified Ag nanoparticles, and found that only the bare (unmodified) nanoparticles were able to prevent virus infection. For comparison, TiO2 nanoparticles failed to intercept the virus. Proteins and lipids may adsorb to nanoparticles forming a so-called bio-corona; however, Ag nanoparticles pre-incubated with pulmonary surfactant retained their ability to block virus infection in the present model. Furthermore, the secondary structure of the spike protein of SARS-CoV-2 was perturbed by the Ag nanoparticles, but not by the ionic control (AgNO3) nor by the TiO2 nanoparticles. Finally, Ag nanoparticles were shown to be non-cytotoxic towards the human lung epithelial cell line BEAS-2B and this was confirmed by using primary human nasal epithelial cells. These results further support that Ag nanoparticles may find use as anti-viral agents.

Understanding the immunological interactions of engineered nanomaterials: Role of the bio-corona.

Engineered nanomaterials are a broad class of materials with the potential for breakthrough applications in many sectors of society not least in medicine. Consequently, safety assessment of nanomaterials and nano-enabled products with respect to human health and the environment is of key importance. To this end, the biological interactions of nanoscale materials must be understood. Here, the dual "identities" of nanomaterials, namely, the material-intrinsic properties or synthetic identity and the acquired, context-dependent properties or biological identity, are discussed in relation to nanomaterial interactions with the immune system, our main defense against foreign intrusion. Specifically, we address whether macrophages and other innate immune cells respond to the synthetic identity or the biological identity of nanomaterials, that is, the surface adsorbed proteins and/or other biomolecules known as the bio-corona, or both? This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials.

Nanomaterial characterization: Understanding nano-bio interactions.

Nanomaterials are marvelously small, yet they may be harnessed to great effect. Much has been learned with respect to the biological interactions and effects of nanomaterials during the past decade. However, characterization of nanomaterials is typically performed on as-synthesized materials. We posit that nanomaterials are dynamic entities and should be studied and regulated as such. Hence, characterization of nanomaterials should take into account their biotransformation. However, in situ characterization of nanomaterials as they undergo dynamic changes (coronation, dissolution, degradation) in a living system remains a formidable challenge in nanosafety. Material scientists and toxicologists need to join forces to address this issue.

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.

Biomarkers of nanomaterials hazard from multi-layer data.

There is an urgent need to apply effective, data-driven approaches to reliably predict engineered nanomaterial (ENM) toxicity. Here we introduce a predictive computational framework based on the molecular and phenotypic effects of a large panel of ENMs across multiple in vitro and in vivo models. Our methodology allows for the grouping of ENMs based on multi-omics approaches combined with robust toxicity tests. Importantly, we identify mRNA-based toxicity markers and extensively replicate them in multiple independent datasets. We find that models based on combinations of omics-derived features and material intrinsic properties display significantly improved predictive accuracy as compared to physicochemical properties alone.