Aerosol exposure at air-liquid-interface (AE-ALI) in vitro toxicity system characterisation: Particle deposition and the importance of air control responses.

Experimental systems allowing aerosol exposure (AE) of cell cultures at the air-liquid-interface (ALI) are increasingly being used to assess the toxicity of inhaled contaminants as they are more biomimetic than standard methods using submerged cultures, however, they require detailed characterisation before use. An AE-ALI system combining aerosol generation with a CULTEX® exposure chamber was characterised with respect to particle deposition and the cellular effects of filtered air (typical control) exposures. The effect of system parameters (electrostatic precipitator voltage, air flowrate to cells and insert size) on deposition efficiency and spatial distribution were investigated using ICP-MS and laser ablation ICP-MS, for an aerosol of CeO2 nanoparticles. Deposition varied with conditions, but appropriate choice of operating parameters produced broadly uniform deposition at suitable levels. The impact of air exposure duration on alveolar cells (A549) and primary small airway epithelial cells (SAECs) was explored with respect to LDH release and expression of selected genes. Results indicated that air exposures could have a significant impact on cells (e.g., cytotoxicity and expression of genes, including CXCL1, HMOX1, and SPP1) at relatively short durations (from 10 mins) and that SAECs were more sensitive. These findings indicate that detailed system characterisation is essential to ensure meaningful results.

Roadmap towards safe and sustainable advanced and innovative materials. (Outlook for 2024-2030).

The adoption of innovative advanced materials holds vast potential, contingent upon addressing safety and sustainability concerns. The European Commission advocates the integration of Safe and Sustainable by Design (SSbD) principles early in the innovation process to streamline market introduction and mitigate costs. Within this framework, encompassing ecological, social, and economic factors is paramount. The NanoSafety Cluster (NSC) delineates key safety and sustainability areas, pinpointing unresolved issues and research gaps to steer the development of safe(r) materials. Leveraging FAIR data management and integration, alongside the alignment of regulatory aspects, fosters informed decision-making and innovation. Integrating circularity and sustainability mandates clear guidance, ensuring responsible innovation at every stage. Collaboration among stakeholders, anticipation of regulatory demands, and a commitment to sustainability are pivotal for translating SSbD into tangible advancements. Harmonizing standards and test guidelines, along with regulatory preparedness through an exchange platform, is imperative for governance and market readiness. By adhering to these principles, the effective and sustainable deployment of innovative materials can be realized, propelling positive transformation and societal acceptance.

A review on nanomaterial-based SERS substrates for sustainable agriculture.

The agricultural sector plays a pivotal role in driving the economy of many developing countries. Any dent in this economical structure may have a severe impact on a country's population. With rising climate change and increasing pollution, the agricultural sector is experiencing significant damage. Over time this cumulative damage will affect the integrity of food crops and create food security issues around the world. Therefore, an early warning system is needed to detect possible stress on food crops. Here we present a review of the recent developments in nanomaterial-based Surface Enhanced Raman Spectroscopy (SERS) substrates which could be utilized to monitor agricultural crop responses to natural and anthropogenic stress. Initially, our review delves into diverse and cost-effective strategies for fabricating SERS substrates, emphasizing their intelligent utilization across various agricultural scenarios. In the second phase of our review, we spotlight the specific application of SERS in addressing critical food security issues. By detecting nutrients, hormones, and effector molecules in plants, SERS provides valuable insights into plant health. Furthermore, our exploration extends to the detection of contaminants, chemicals, and foodborne pathogens within plants, showcasing the versatility of SERS in ensuring food safety. The cumulative knowledge derived from these discussions illustrates the transformative potential of SERS in bolstering the agricultural economy. By enhancing precision in nutrient management, monitoring plant health, and enabling rapid detection of harmful substances, SERS emerges as a pivotal tool in promoting sustainable and secure agricultural practices. Its integration into agricultural processes not only augments productivity but also establishes a robust defence against potential threats to crop yield and food quality. As SERS continues to evolve, its role in shaping the future of agriculture becomes increasingly pronounced, promising a paradigm shift in how we approach and address challenges in food production and safety.

Modulation of Heat Shock Protein Expression in Alveolar Adenocarcinoma Cells through Gold Nanoparticles and Cisplatin Treatment.

Heat-shock proteins (HSPs) are stress-responsive molecules belonging to the family of evolutionary molecular chaperones known to be crucial in many cancer types, including human alveolar adenocarcinoma cells (A549). These proteins are highly overexpressed in cancers to support their ability to accommodate imbalances in cell signalling, DNA alterations, proteins, and energy metabolism associated with oncogenesis. The current study evaluated the effects of gold nanoparticles (AuNPs) combined with cisplatin (CDDP) on molecular chaperone HSPs in A549 cells. It was found that AuNPs:CDDP decreased the percentage of cell viability (38.5%) measured using the modified lactated dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. AuNPs:CDDP exposure caused a significant (p < 0.05) increase in reactive oxygen species (ROS) generation by 1.81-fold, apoptosis induction, and a decrease in the mitochondrial membrane potential (MMP) compared to AuNPs or CDDP alone. Similarly, exposure to the AuNPs:CDDP combination had pronounced cytotoxic effects on the expression of HSPs and PI3K/AKT/mTOR, as well as apoptosis-related proteins. The results demonstrate that the combination of AuNPs with CDDP might enhance the anticancer efficacy of CDDP.

Learning from Safe-by-Design for Safe-and-Sustainable-by-Design: Mapping the current landscape of Safe-by-Design reviews, case studies, and frameworks.

With the introduction of the European Commission's "Safe and Sustainable-by-Design" (SSbD) framework, the interest in understanding the implications of safety and sustainability assessments of chemicals, materials, and processes at early-innovation stages has skyrocketed. Our study focuses on the "Safe-by-Design" (SbD) approach from the nanomaterials sector, which predates the SSbD framework. In this assessment, SbD studies have been compiled and categorized into reviews, case studies, and frameworks. Reviews of SbD tools have been further classified as quantitative, qualitative, or toolboxes and repositories. We assessed the SbD case studies and classified them into three categories: safe(r)-by-modeling, safe(r)-by-selection, or safe(r)-by-redesign. This classification enabled us to understand past SbD work and subsequently use it to define future SSbD work so as to avoid confusion and possibilities of "SSbD-washing" (similar to greenwashing). Finally, the preexisting SbD frameworks have been studied and contextualized against the SSbD framework. Several key recommendations for SSbD based on our analysis can be made. Knowledge gained from existing approaches such as SbD, green and sustainable chemistry, and benign-by-design approaches needs to be preserved and effectively transferred to SSbD. Better incorporation of chemical and material functionality into the SSbD framework is required. The concept of lifecycle thinking and the stage-gate innovation model need to be reconciled for SSbD. The development of high-throughput screening models is critical for the operationalization of SSbD. We conclude that the rapid pace of both SbD and SSbD development necessitates a regular mapping of the newly published literature that is relevant to this field.

Functionalized Gold Nanoparticles Suppress the Proliferation of Human Lung Alveolar Adenocarcinoma Cells by Deubiquitinating Enzymes Inhibition.

Functionalized gold nanoparticles (AuNPs) are widely used in therapeutic applications, but little is known regarding the impact of their surface functionalization in the process of toxicity against cancer cells. This study investigates the anticancer effects of 5 nm spherical AuNPs functionalized with tannate, citrate, and PVP on deubiquitinating enzymes (DUBs) in human lung alveolar adenocarcinoma (A549) cells. Our findings show that functionalized AuNPs reduce the cell viability in a concentration- and time-dependent manner as measured by modified lactate dehydrogenase (mLDH) and 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. An increased generation of intracellular reactive oxygen species (ROS) and depletion of glutathione (GSH/GSSG) ratio was observed with the highest AuNP concentration of 10 µg/mL. The expression of DUBs such as ubiquitin specific proteases (USP7, USP8, and USP10) was slightly inhibited when treated with concentrations above 2.5 µg/mL. Moreover, functionalized AuNPs showed an inhibitory effect on protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) and wingless-related integration site (Wnt) signaling proteins, and this could further trigger mitochondrial related-apoptosis by the upregulation of caspase-3, caspase-9, and PARP in A549 cells. Furthermore, our study shows a mechanistic understanding of how functionalized AuNPs inhibit the DUBs, consequently suppressing cell proliferation, and can be modulated as an approach toward anticancer therapy. The study also warrants the need for future work to investigate the effect of functionalized AuNPs on DUB on other cancer cell lines both in vitro and in vivo.

A Review of the Aquatic Environmental Transformations of Engineered Nanomaterials.

Once released into the environment, engineered nanomaterials (ENMs) undergo complex interactions and transformations that determine their fate, exposure concentration, form, and likely impact on biota. Transformations are physical, chemical, or biological changes that occur to the ENM or the ENM coating. Over time, these transformations have an impact on their behaviour and properties. The interactions and transformations of ENMs in the environment depend on their pristine physical and chemical characteristics and the environmental or biological compartment into which they are released. The uniqueness of each ENM property or lifecycle results in a great deal of complexity. Even small changes may have a significant impact on their potential transformations. This review outlines the key influences and outcomes of ENM evolution pathways in aquatic environments and provides an assessment of potential environmental transformations, focusing on key chemical, physical, and biological processes. By obtaining a comprehensive understanding of the potential environmental transformations that nanomaterials can undergo, more realistic models of their probable environmental behaviour and potential impact can be developed. This will, in turn, be crucial in supporting regulatory bodies in their efforts to develop environmental policy in the field of nanotechnology.

Nanotoxicity of Graphene Oxide - Gold Nanohybrid to Daphnia magna.

The expanding use of hybrid nanomaterials in many applications necessitates evaluation of their environmental risks. This study investigates the acute toxicity and bioaccumulation of graphene oxide - gold (GO-Au) nanohybrid in neonates (<24 hrs old) of Daphnia magna after exposure to a wide range of concentrations (1-100 mg/L). No significant mortality or immobilisation was observed after the exposure period. Microscopic observation showed an uptake of the nanohybrid and internal damage in the gut of the exposed organisms. Bioaccumulation of the GO-Au nanohybrid also occurred in a concentration-dependant manner. Continuous evaluation of the environmental risks from exposure to this nanohybrid and other advanced materials is imperative to avert disruption to the ecosystem.

Gold Nanoparticles Induced Size Dependent Cytotoxicity on Human Alveolar Adenocarcinoma Cells by Inhibiting the Ubiquitin Proteasome System.

Gold nanoparticles (AuNPs) are widely used in biomedicine due to their remarkable therapeutic applications. However, little is known about their cytotoxic effects on the ubiquitin proteasome system (UPS). Herein, the cytotoxicity of different sizes of AuNPs (5, 10, and 80 nm) on the UPS was investigated with a particular focus on deubiquitinating enzymes (DUBs) such as ubiquitin-specific proteases (USP) and ubiquitin carboxyl-terminal hydrolases (UCHL-1) in human alveolar epithelial adenocarcinoma (A549). It was found that all sizes of AuNPs reduced the percentage of viable A549 cells and increased lactate dehydrogenase (LDH) release, measured using the MTT and LDH assays, respectively. Furthermore, the 5 nm AuNPs were found to exhibit greater cytotoxicity than the 10 and 80 nm AuNPs. In addition, apoptosis and necrosis were activated through reactive oxygen species (ROS) generation due to AuNPs exposure. The internalisation of AuNPs in A549 cells increased with increasing particle size (80 > 10 > 5 nm). Interestingly, the expression of USP7, USP8, USP10, and UCHL-1 was significantly (p < 0.001) downregulated upon treatment with 5-30 µg/mL of all the AuNPs sizes compared to control cells. Moreover, the inhibition of these proteins triggered mitochondrial-related apoptosis through the upregulation of poly (ADP-ribose) polymerase (PARP), caspase-3, and caspase-9. Collectively, these results indicate that AuNPs suppress the proliferation of A549 cells and can potentially be used as novel inhibitors of the proteasome.

Submicron Plastic Adsorption by Peat, Accumulation in Sphagnum Mosses and Influence on Bacterial Communities in Peatland Ecosystems.

The smallest fraction of plastic pollution, submicron plastics (SMPs <1 µm) are expected to be ubiquitous in the environment. No information is available about SMPs in peatlands, which have a key role in sequestering carbon in terrestrial ecosystems. It is unknown how these plastic particles might behave and interact with (micro)organisms in these ecosystems. Here, we show that the chemical composition of polystyrene (PS) and poly(vinyl chloride) (PVC)-SMPs influenced their adsorption to peat. Consequently, this influenced the accumualtion of SMPs by Sphagnum moss and the composition and diversity of the microbial communities in peatland. Natural organic matter (NOM), which adsorbs from the surrounding water to the surface of SMPs, decreased the adsorption of the particles to peat and their accumulation by Sphagnum moss. However, the presence of NOM on SMPs significantly altered the bacterial community structure compared to SMPs without NOM. Our findings show that peatland ecosystems can potentially adsorb plastic particles. This can not only impact mosses themselves but also change the local microbial communities.

Application of Isotopically Labeled Engineered Nanomaterials for Detection and Quantification in Soils via Single-Particle Inductively Coupled Plasma Time-of-Flight Mass Spectrometry.

Finding and quantifying engineered nanomaterials (ENMs) in soil are challenging because of the abundance of natural nanomaterials (NNMs) with the same elemental composition, for example, TiO2. Isotopically enriched ENMs may be distinguished from NNMs with the same elemental composition using single-particle inductively coupled plasma time-of-flight mass spectrometry (spICP-TOF-MS) to measure multiple isotopes simultaneously within each ENM and NNM in soil, but the minimum isotope enrichment needed for detection of ENMs in soil is not known. Here, we determined the isotope enrichment needed for 47Ti-enriched TiO2 ENMs to be detectable in soil and assessed the effects of weathering on those requirements for less soluble TiO2 and more soluble CuO ENMs. The isotope-enriched ENMs were dosed into two different soils and were extracted and measured by spICP-TOF-MS after 1, 7, and 30 days. Isotope-enriched ENMs were recovered and detected for all three time points. The 47Ti-enriched TiO2 ENMs were detectable in Lufa 2.2 soil at a nominal dosed concentration of 10 mg-TiO2 kg-1 which is an environmentally relevant concentration in biosolid-amended soils. For distinguishing an ∼70 nm diameter TiO2 ENM from TiO2 NNMs in Lufa 2.2 soil, an ∼10 wt % 47Ti isotope-enrichment was required, and this enrichment requirement increases as the particle size decreases. This study is the first to evaluate the tracking ability of isotope-enriched ENMs at an individual particle level in soil and provides guidance on the isotope enrichment requirements for quantification of ENMs made from Earth-abundant elements in soils.

Correction: Peters et al. Benchmarking the ACEnano Toolbox for Characterisation of Nanoparticle Size and Concentration by Interlaboratory Comparison. Molecules 2021, 26, 5315.

Due to miscommunication a number of potential co-authors were not listed in the original publication [...].

Representing and describing nanomaterials in predictive nanoinformatics.

Engineered nanomaterials (ENMs) enable new and enhanced products and devices in which matter can be controlled at a near-atomic scale (in the range of 1 to 100 nm). However, the unique nanoscale properties that make ENMs attractive may result in as yet poorly known risks to human health and the environment. Thus, new ENMs should be designed in line with the idea of safe-and-sustainable-by-design (SSbD). The biological activity of ENMs is closely related to their physicochemical characteristics, changes in these characteristics may therefore cause changes in the ENMs activity. In this sense, a set of physicochemical characteristics (for example, chemical composition, crystal structure, size, shape, surface structure) creates a unique 'representation' of a given ENM. The usability of these characteristics or nanomaterial descriptors (nanodescriptors) in nanoinformatics methods such as quantitative structure-activity/property relationship (QSAR/QSPR) models, provides exciting opportunities to optimize ENMs at the design stage by improving their functionality and minimizing unforeseen health/environmental hazards. A computational screening of possible versions of novel ENMs would return optimal nanostructures and manage ('design out') hazardous features at the earliest possible manufacturing step. Safe adoption of ENMs on a vast scale will depend on the successful integration of the entire bulk of nanodescriptors extracted experimentally with data from theoretical and computational models. This Review discusses directions for developing appropriate nanomaterial representations and related nanodescriptors to enhance the reliability of computational modelling utilized in designing safer and more sustainable ENMs.

Reduced synaptic activity and dysregulated extracellular matrix pathways in midbrain neurons from Parkinson's disease patients.

Several mutations that cause Parkinson's disease (PD) have been identified over the past decade. These account for 15-25% of PD cases; the rest of the cases are considered sporadic. Currently, it is accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from the induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, show changes that are central and convergent to PD and suggest a strong involvement of the tetra-partite synapse in PD pathophysiology.

Toxicological Impact and in Vivo Tracing of Rhodamine Functionalised ZIF-8 Nanoparticles.

Metal Organic Frameworks (MOFs) are extensively used for a wide range of applications due to their exceptionally high surface area. MOF particles are conventionally in micron size, but the nanosized MOFs show good transportation/mobility due to their small size, and when combined with the high surface area of MOFs, it makes MOF nanoparticles an ideal candidate to study for environmental remediation. Therefore, it is important to study the ecotoxicological impact of these MOFs. In this study, we developed rhodamine labelled nanoparticles of zinc imidazolate metal organic framework (ZIF-8 MOFs) as a means of in vivo tracing the MOF translocation in C. elegans. Rhodamine B isothiocyanate functionalized ZIF-8 MOFs nanoparticles (RBITC@ZIF-8 MOF nanoparticles; size 44 ± 7 nm) were fed to the worms naturally within a concentration range of 0.16-16.4 µg mg-1. Fluorescence was detected in the pharyngeal and gut lumen regions of the worms after 4 h of treatment, for exposure concentrations >0.163 µg mg-1. A higher intensity of fluorescence was observed at the end of 24 h for all exposure concentrations. Worms treated with RBITC@ZIF-8 MOF concentrations of ≥1.63 µg mg-1 for 24 h showed a bright stable fluorescence signal at the tail region. The uptake of RBITC@ZIF-8 MOF for an exposure concentration of 0.163, 1.63, and 8.2 µg mg-1 was found to be 52.1, 11.4 and 28.6%, respectively. Through this study, we showed that RBITC@ZIF-8 MOFs can be exposed to C. elegans and imaged at low concentrations of ∼0.16 µg mg-1.

Effects of nanopolystyrene addition on nitrogen fertilizer fate, gaseous loss of N from the soil, and soil microbial community composition.

Nanoplastics and microplastics are the degradation products of plastics waste and have become a dominant pollutant in the environment. However, little is known about the ecological impacts of nanoplastic particles in the agroecosystem. We conducted a mesocosm experiment to examine nanopolystyrene effects on fertilizer nitrogen (N) fate, N gaseous losses and soil microbial communities using Chinese cabbage (Brassica Campestris ssp.) as the model plant. The two-factorial experiment was designed as the addition of 15N-labeled urea exposed without and with ~50 nm nanopolystyrene (0, 0.05%, and 0.1%). Nanopolystyrene addition had a detectable effect on soil mineral N content. The 15N uptake of plants was reduced in aboveground biomass but enhanced in roots with increasing nanopolystyrene concentration. Nanopolystyrene addition decreased soil nitrous oxide and ammonia emissions by 27% and 37%, respectively. Nanopolystyrene addition consistently reduced the abundance of ammonia oxidizer genes but showed contrasting effects on denitrifying genes. Metagenomic sequencing data revealed no significant effects of nanopolystyrene on the N-cycle pathway, while it significantly altered the composition of bacterial and fungal communities. This study provided the first insights into the nanopolystyrene induced linkage of root growth with more root N uptake and less gaseous N losses and the associated changes in the microbial community.

An analytical workflow for dynamic characterization and quantification of metal-bearing nanomaterials in biological matrices.

To assess the safety of engineered nanomaterials (ENMs) and to evaluate and improve ENMs' targeting ability for medical application, it is necessary to analyze the fate of these materials in biological media. This protocol presents a workflow that allows researchers to determine, characterize and quantify metal-bearing ENMs (M-ENMs) in biological tissues and cells and quantify their dynamic behavior at trace-level concentrations. Sample preparation methods to enable analysis of M-ENMs in a single cell, a cell layer, tissue, organ and physiological media (e.g., blood, gut content, hemolymph) of different (micro)organisms, e.g., bacteria, animals and plants are presented. The samples are then evaluated using fit-for-purpose analytical techniques e.g., single-cell inductively coupled plasma mass spectrometry, single-particle inductively coupled plasma mass spectrometry and synchrotron X-ray absorption fine structure, providing a protocol that allows comprehensive characterization and quantification of M-ENMs in biological matrices. Unlike previous methods, the protocol uses no fluorescent dyes or radiolabels to trace M-ENMs in biota and enables analysis of most M-ENMs at cellular, tissue and organism levels. The protocols can be applied by a wide variety of users depending on the intended purpose of the application, e.g., to correlate toxicity with a specific particle form, or to understand the absorption, distribution and excretion of M-ENMs. The results facilitate an understanding of the biological fate of M-ENMs and their dynamic behavior in biota. Performing the protocol may take 7-30 d, depending on which combination of methods is applied.

Automation and Standardization-A Coupled Approach towards Reproducible Sample Preparation Protocols for Nanomaterial Analysis.

Whereas the characterization of nanomaterials using different analytical techniques is often highly automated and standardized, the sample preparation that precedes it causes a bottleneck in nanomaterial analysis as it is performed manually. Usually, this pretreatment depends on the skills and experience of the analysts. Furthermore, adequate reporting of the sample preparation is often missing. In this overview, some solutions for techniques widely used in nano-analytics to overcome this problem are discussed. Two examples of sample preparation optimization by automation are presented, which demonstrate that this approach is leading to increased analytical confidence. Our first example is motivated by the need to exclude human bias and focuses on the development of automation in sample introduction. To this end, a robotic system has been developed, which can prepare stable and homogeneous nanomaterial suspensions amenable to a variety of well-established analytical methods, such as dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), field-flow fractionation (FFF) or single-particle inductively coupled mass spectrometry (sp-ICP-MS). Our second example addresses biological samples, such as cells exposed to nanomaterials, which are still challenging for reliable analysis. An air-liquid interface has been developed for the exposure of biological samples to nanomaterial-containing aerosols. The system exposes transmission electron microscopy (TEM) grids under reproducible conditions, whilst also allowing characterization of aerosol composition with mass spectrometry. Such an approach enables correlative measurements combining biological with physicochemical analysis. These case studies demonstrate that standardization and automation of sample preparation setups, combined with appropriate measurement processes and data reduction are crucial steps towards more reliable and reproducible data.

Correction: Quevedo et al. Mechanisms of Silver Nanoparticle Uptake by Embryonic Zebrafish Cells. Nanomaterials 2021, 11, 2699.

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