Current Challenges to Align Inflammatory Key Events in Animals and Lung Cell Models In Vitro.

With numerous novel and innovative in vitro models emerging every year to reduce or replace animal testing, there is an urgent need to align the design, harmonization, and validation of such systems using in vitro-in vivo extrapolation (IVIVE) approaches. In particular, in inhalation toxicology, there is a lack of predictive and prevalidated in vitro lung models that can be considered a valid alternative for animal testing. The predictive power of such models can be enhanced by applying the Adverse Outcome Pathways (AOP) framework, which casually links key events (KE) relevant to IVIVE. However, one of the difficulties identified is that the endpoint analysis and readouts of specific assays in in vitro and animal models for specific toxicants are currently not harmonized, making the alignment challenging. We summarize the current state of the art in endpoint analysis in the two systems, focusing on inflammatory-induced effects and providing guidance for future research directions to improve the alignment.

Theoretical evaluation of the impact of diverse treatment conditions by calculation of the tumor control probability (TCP) of simulated cervical cancer Hyperthermia-Radiotherapy (HT-RT) treatments in-silico.

INTRODUCTION: Hyperthermia (HT) induces various cellular biological processes, such as repair impairment and direct HT cell killing. In this context, in-silico biophysical models that translate deviations in the treatment conditions into clinical outcome variations may be used to study the extent of such processes and their influence on combined hyperthermia plus radiotherapy (HT + RT) treatments under varying conditions. METHODS: An extended linear-quadratic model calibrated for SiHa and HeLa cell lines (cervical cancer) was used to theoretically study the impact of varying HT treatment conditions on radiosensitization and direct HT cell killing effect. Simulated patients were generated to compute the Tumor Control Probability (TCP) under different HT conditions (number of HT sessions, temperature and time interval), which were randomly selected within margins based on reported patient data. RESULTS: Under the studied conditions, model-based simulations suggested a treatment improvement with a total CEM43 thermal dose of approximately 10 min. Additionally, for a given thermal dose, TCP increased with the number of HT sessions. Furthermore, in the simulations, we showed that the TCP dependence on the temperature/time interval is more correlated with the mean value than with the minimum/maximum value and that comparing the treatment outcome with the mean temperature can be an excellent strategy for studying the time interval effect. CONCLUSION: The use of thermoradiobiological models allows us to theoretically study the impact of varying thermal conditions on HT + RT treatment outcomes. This approach can be used to optimize HT treatments, design clinical trials, and interpret patient data.

Combining analytical techniques to assess the translocation of diesel particles across an alveolar tissue barrier in vitro.

BACKGROUND: During inhalation, airborne particles such as particulate matter ≤ 2.5 µm (PM2.5), can deposit and accumulate on the alveolar epithelial tissue. In vivo studies have shown that fractions of PM2.5 can cross the alveolar epithelium to blood circulation, reaching secondary organs beyond the lungs. However, approaches to quantify the translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, methods to assess the translocation of standard diesel exhaust particles (DEPs) across permeable polyethylene terephthalate (PET) inserts at 0.4, 1, and 3 µm pore sizes were first optimized with transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-VIS), and lock-in thermography (LIT), which were then applied to study the translocation of DEPs across human alveolar epithelial type II (A549) cells. A549 cells that grew on the membrane (pore size: 3 µm) in inserts were exposed to DEPs at different concentrations from 0 to 80 µg.mL- 1 ( 0 to 44 µg.cm- 2) for 24 h. After exposure, the basal fraction was collected and then analyzed by combining qualitative (TEM) and quantitative (UV-VIS and LIT) techniques to assess the translocated fraction of the DEPs across the alveolar epithelium in vitro. RESULTS: We could detect the translocated fraction of DEPs across the PET membranes with 3 µm pore sizes and without cells by TEM analysis, and determine the percentage of translocation at approximatively 37% by UV-VIS (LOD: 1.92 µg.mL- 1) and 75% by LIT (LOD: 0.20 µg.cm- 2). In the presence of cells, the percentage of DEPs translocation across the alveolar tissue was determined around 1% at 20 and 40 µg.mL- 1 (11 and 22 µg.cm- 2), and no particles were detected at higher and lower concentrations. Interestingly, simultaneous exposure of A549 cells to DEPs and EDTA can increase the translocation of DEPs in the basal fraction. CONCLUSION: We propose a combination of analytical techniques to assess the translocation of DEPs across lung tissues. Our results reveal a low percentage of translocation of DEPs across alveolar epithelial tissue in vitro and they correspond to in vivo findings. The combination approach can be applied to any traffic-generated particles, thus enabling us to understand their involvement in public health.

Understanding The Benefits and Risks of Sustainable Nanomaterials in a Research Environment.

Nanomaterials hold immense potential for numerous applications in energy, health care, and environmental sectors, playing an important role in our daily lives. Their utilization spans from improving energy efficiency to enhancing medical diagnostics, and mitigating environmental pollution, thus presenting a multifaceted approach towards achieving sustainability goals. To ensure the sustainable and safe utilization of nanomaterials, a thorough evaluation of potential hazards and risks is essential throughout their lifecycle-from resource extraction and production to use and disposal. In this review, we focus on understanding and addressing potential environmental and health risks associated with nanomaterial utilization. We advocate for a balanced approach with early hazard identification, safe-by-design principles, and life cycle assessments, while emphasizing safe handling and disposal practices, collaboration, and continuous improvement. Our goal is to ensure responsible nanotechnology development, fostering innovation alongside environmental and community well-being, through a holistic approach integrating science, ethics, and proactive risk assessment.

The Influence of Liquid Menisci on Nanoparticle Dosimetry in Submerged Cells.

Understanding the interaction between cells and nanoparticles (NPs) is vital to understand the hazard associated with nanoparticles. This requires quantifying and interpreting dose-response relationships. Experiments with cells cultured in vitro and exposed to particle dispersions mainly rely on mathematical models that estimate the received nanoparticle dose. However, models need to consider that aqueous cell culture media wets the inner surface of hydrophilic open wells, which results in a curved liquid-air interface called the meniscus. Here the impact of the meniscus on nanoparticle dosimetry is addressed in detail. Experiments and build an advanced mathematical model, to demonstrate that the presence of the meniscus may bring about systematic errors that must be considered to advance reproducibility and harmonization is presented. The script of the model is co-published and can be adapted to any experimental setup. Finally, simple and practical solutions to this problem, such as covering the air-liquid interface with a permeable lid or soft rocking of the cell culture well plate is proposed.

Correlative Light, Electron Microscopy and Raman Spectroscopy Workflow To Detect and Observe Microplastic Interactions with Whole Jellyfish.

Many researchers have turned their attention to understanding microplastic interaction with marine fauna. Efforts are being made to monitor exposure pathways and concentrations and to assess the impact such interactions may have. To answer these questions, it is important to select appropriate experimental parameters and analytical protocols. This study focuses on medusae of Cassiopea andromeda jellyfish: a unique benthic jellyfish known to favor (sub-)tropical coastal regions which are potentially exposed to plastic waste from land-based sources. Juvenile medusae were exposed to fluorescent poly(ethylene terephthalate) and polypropylene microplastics (<300 µm), resin embedded, and sectioned before analysis with confocal laser scanning microscopy as well as transmission electron microscopy and Raman spectroscopy. Results show that the fluorescent microplastics were stable enough to be detected with the optimized analytical protocol presented and that their observed interaction with medusae occurs in a manner which is likely driven by the microplastic properties (e.g., density and hydrophobicity).

Pitfalls in methods to study colocalization of nanoparticles in mouse macrophage lysosomes.

BACKGROUND: In the field of nanoscience there is an increasing interest to follow dynamics of nanoparticles (NP) in cells with an emphasis on endo-lysosomal pathways and long-term NP fate. During our research on this topic, we encountered several pitfalls, which can bias the experimental outcome. We address some of these pitfalls and suggest possible solutions. The accuracy of fluorescence microscopy methods has an important role in obtaining insights into NP interactions with lysosomes at the single cell level including quantification of NP uptake in a specific cell type. METHODS: Here we use J774A.1 cells as a model for professional phagocytes. We expose them to fluorescently-labelled amorphous silica NP with different sizes and quantify the colocalization of fluorescently-labelled NP with lysosomes over time. We focus on confocal laser scanning microscopy (CLSM) to obtain 3D spatial information and follow live cell imaging to study NP colocalization with lysosomes. RESULTS: We evaluate different experimental parameters that can bias the colocalization coefficients (i.e., Pearson's and Manders'), such as the interference of phenol red in the cell culture medium with the fluorescence intensity and image post-processing (effect of spatial resolution, optical slice thickness, pixel saturation and bit depth). Additionally, we determine the correlation coefficients for NP entering the lysosomes under four different experimental set-ups. First, we found out that not only Pearson's, but also Manders' correlation coefficient should be considered in lysosome-NP colocalization studies; second, there is a difference in NP colocalization when using NP of different sizes and fluorescence dyes and last, the correlation coefficients might change depending on live-cell and fixed-cell imaging set-up. CONCLUSIONS: The results summarize detailed steps and recommendations for the experimental design, staining, sample preparation and imaging to improve the reproducibility of colocalization studies between the NP and lysosomes.

Understanding nanoparticle endocytosis to improve targeting strategies in nanomedicine.

Nanoparticles (NPs) have attracted considerable attention in various fields, such as cosmetics, the food industry, material design, and nanomedicine. In particular, the fast-moving field of nanomedicine takes advantage of features of NPs for the detection and treatment of different types of cancer, fibrosis, inflammation, arthritis as well as neurodegenerative and gastrointestinal diseases. To this end, a detailed understanding of the NP uptake mechanisms by cells and intracellular localization is essential for safe and efficient therapeutic applications. In the first part of this review, we describe the several endocytic pathways involved in the internalization of NPs and we discuss the impact of the physicochemical properties of NPs on this process. In addition, the potential challenges of using various inhibitors, endocytic markers and genetic approaches to study endocytosis are addressed along with the principal (semi) quantification methods of NP uptake. The second part focuses on synthetic and bio-inspired substances, which can stimulate or decrease the cellular uptake of NPs. This approach could be interesting in nanomedicine where a high accumulation of drugs in the target cells is desirable and clearance by immune cells is to be avoided. This review contributes to an improved understanding of NP endocytic pathways and reveals potential substances, which can be used in nanomedicine to improve NP delivery.

Understanding the Development, Standardization, and Validation Process of Alternative In Vitro Test Methods for Regulatory Approval from a Researcher Perspective.

Due to economic, practical, ethical, and scientific reasons, researchers, among others, are pushing for alternative in vitro test methods to replace or reduce existing animal experiments. In order for these tests to be more broadly used by the industrial sector and regulatory bodies, orchestrated efforts are required to show the robustness and reliability of in vitro methods, which can accelerate the use for early screening testing. Another way of increasing the use of alternatives is to coordinate validation studies, that is, multi-laboratory trials, and to gain regulatory approval and instatement as test guidelines or standard method. However, awareness of the exact standardization, validation, and approval process has been a major obstacle for many researchers. Herein, the process has been broken down into three main phases: i) test method development; ii) intra- and inter-laboratory validation; and iii) regulatory acceptance. This general process applies to all alternative methods seeking validation and approval, although the intricacies of different toxicological endpoints and/or chemical sectors may lead to additional work, particularly in the validation stage. The authors' aim is to provide insight in the development process of alternative methods with a focus on in vitro cell culture methods over validation to regulatory acceptance.

Additional Commentary on the Detection and Quantification of Plastic Micro- and Nanoparticles in Tea Samples.

The study of plastic particles, particularly those in the micro-, sub-micro-, and nano-size ranges, within food and beverages has gained increasing interest within recent years. However, many analytical techniques have limits of detection which hinder their use for the study of these particles in these sample matrices. In addition, remaining contaminants from the matrices can interfere with the signals from plastic particles. Thus, great care must be given to sample preparation and data interpretation to ensure accurate results. This study proposes the use of sample purification through chemical digestion protocols to facilitate the study of plastic particles present in tea samples, and serves to highlight technical limitations which must be overcome in future studies.

Resolution Limit of Taylor Dispersion: An Exact Theoretical Study.

Taylor dispersion is a microfluidic analytical technique with a high dynamic range and therefore is suited well to measuring the hydrodynamic radius of small molecules, proteins, supramolecular complexes, macromolecules, nanoparticles and their self-assembly. Here we calculate an unaddressed yet fundamental property: the limit of resolution, which is defined as the smallest change in the hydrodynamic radius that Taylor dispersion can resolve accurately and precisely. Using concepts of probability theory and inferential statistics, we present a comprehensive theoretical approach, addressing uniform and polydisperise particle systems, which involve either model-based or numerical analyses. We find a straightforward scaling relationship in which the resolution limit is linearly proportional to the optical-extinction-weighted average hydrodynamic radius of the particle systems.

Versatile Macroscale Concentration Gradients of Nanoparticles in Soft Nanocomposites.

Nanocomposite materials benefit from the diverse physicochemical properties featured by nanoparticles, and the presence of nanoparticle concentration gradients can lend functions to macroscopic materials beyond the realm of classical nanocomposites. It is shown here that linearity and time-shift invariance obtained via the synergism of two independent physical phenomena-translational self-diffusion and shear-driven dispersion-may give access to an exceptionally high degree of flexibility in the design of scalable and programmable long-range concentration gradients of nanoparticles in solidifiable liquid matrices.

Impact of airborne particulate matter on skin: a systematic review from epidemiology to in vitro studies.

BACKGROUND: Air pollution is killing close to 5 million people a year, and harming billions more. Air pollution levels remain extremely high in many parts of the world, and air pollution-associated premature deaths have been reported for urbanized areas, particularly linked to the presence of airborne nano-sized and ultrafine particles. MAIN TEXT: To date, most of the research studies did focus on the adverse effects of air pollution on the human cardiovascular and respiratory systems. Although the skin is in direct contact with air pollutants, their damaging effects on the skin are still under investigation. Epidemiological data suggested a correlation between exposure to air pollutants and aggravation of symptoms of chronic immunological skin diseases. In this study, a systematic literature review was conducted to understand the current knowledge on the effects of airborne particulate matter on human skin. It aims at providing a deeper understanding of the interactions between air pollutants and skin to further assess their potential risks for human health. CONCLUSION: Particulate matter was shown to induce a skin barrier dysfunction and provoke the formation of reactive oxygen species through direct and indirect mechanisms, leading to oxidative stress and induced activation of the inflammatory cascade in human skin. Moreover, a positive correlation was reported between extrinsic aging and atopic eczema relative risk with increasing particulate matter exposure.

A novel 3D intestine barrier model to study the immune response upon exposure to microplastics.

The plausibility of human exposure to microplastics has increased within the last years. Microplastics have been found in different food types including seafood, salt, sugar and beverages. So far, human health effects of microplastics after ingestion are unknown. Herein, we designed a novel, three-dimensional in vitro intestinal model consisting of the human intestinal epithelial cell lines Caco-2 and HT29-MTX-E12 as well as human blood monocyte-derived macrophages and dendritic cells that is suitable to assess the possible effects of ingested microplastics. Relevant microplastic particles (in the order of 50-500 µm), including polymers representing tire wear and polyolefins, which represent major sources of microplastic in the EU, were compared to other polymer classes and an inorganic microparticle, healing earth, which is intended for human consumption. Microplastic particles were exposed at concentrations of 823.5-1380.0 µg/cm2 to the model using a dry powder insufflator system to aerosolize the particles directly on the intestinal model's surface. Cytotoxicity was investigated after 6, 24 and 48 h of exposure via measuring the release of lactate dehydrogenase. Inflammatory end points including the cytokines IL-8, TNFα and IL-1ß as well as changes of the barrier integrity after exposure were additionally monitored. We demonstrated that all of the microplastics and the healing earth particles did not cause any significant cytotoxicity or release of (pro-)inflammatory cytokines and did not change the barrier integrity of the co-culture at any of the time points investigated.

An In Vitro Lung System to Assess the Proinflammatory Hazard of Carbon Nanotube Aerosols.

In vitro three-dimensional (3D) lung cell models have been thoroughly investigated in recent years and provide a reliable tool to assess the hazard associated with nanomaterials (NMs) released into the air. In this study, a 3D lung co-culture model was optimized to assess the hazard potential of multiwalled carbon nanotubes (MWCNTs), which is known to provoke inflammation and fibrosis, critical adverse outcomes linked to acute and prolonged NM exposure. The lung co-cultures were exposed to MWCNTs at the air-liquid interface (ALI) using the VITROCELL® Cloud system while considering realistic occupational exposure doses. The co-culture model was composed of three human cell lines: alveolar epithelial cells (A549), fibroblasts (MRC-5), and macrophages (differentiated THP-1). The model was exposed to two types of MWCNTs (Mitsui-7 and Nanocyl) at different concentrations (2-10 µg/cm2) to assess the proinflammatory as well as the profibrotic responses after acute (24 h, one exposure) and prolonged (96 h, repeated exposures) exposure cycles. The results showed that acute or prolonged exposure to different concentrations of the tested MWCNTs did not induce cytotoxicity or apparent profibrotic response; however, suggested the onset of proinflammatory response.

Nanoparticles and Taylor Dispersion as a Linear Time-Invariant System.

The physical principles underpinning Taylor dispersion offer a high dynamic range to characterize the hydrodynamic radius of particles. While Taylor dispersion grants the ability to measure radius within nearly 5 orders of magnitude, the detection of particles is never instantaneous. It requires a finite sample volume, a finite detector area, and a finite detection time for measuring absorbance. First we show that these practical requirements bias the analysis when the self-diffusion coefficient of particles is high, which is typically the case of small nanoparticles. Second we show that the accuracy of the technique may be recovered by treating Taylor dispersion as a linear time-invariant system, which we prove by analyzing the Taylor dispersion spectra of two iron-oxide nanoparticles measured under identical experimental conditions. The consequence is that such treatment may be necessary whenever Taylor dispersion analysis is not optimized for a given size but dedicated to characterize broad groups of particles of varying size and material.

Precision of Taylor Dispersion.

Taylor dispersion is capable of measuring accurately the hydrodynamic radius over several orders of magnitude. Accordingly, it is now a highly competitive technique dedicated to characterizing small molecules, proteins, macromolecules, nanoparticles, and their self-assembly. Regardless, an in-depth analysis addressing the precision of the technique, being a key indicator of reproducibility, is not available. Benefiting from analytical modeling and statistical analysis, we address error propagation and present a comprehensive theoretical study of the precision of Taylor dispersion. Theory is then compared against experiment, and we find full consistency. Our results are most helpful when the design, objectives, or control of analytical quality is in focus.

Form Follows Function: Nanoparticle Shape and Its Implications for Nanomedicine.

This review is a comprehensive description of the past decade of research into understanding how the geometry and size of nanoparticles affect their interaction with biological systems: from single cells to whole organisms. Recently, there has been a great deal of effort to use both the shape and the size of nanoparticles to target specific cellular uptake mechanisms, biodistribution patterns, and pharmacokinetics. While the successes of spherical lipid-based nanoparticles have heralded marked changes in chemotherapy worldwide, the history of asbestos-induced lung disease casts a long shadow over fibrous materials to date. The impact of particle morphology is known to be intertwined with many physicochemical parameters, namely, size, elasticity, surface chemistry, and biopersistence. In this review, we first highlight some of the morphologies observed in nature as well as shapes available to us through synthetic strategies. Following this we discuss attempts to understand the cellular uptake of nanoparticles through various theoretical models before comparing this with observations from in vitro and in vivo experiments. In addition, we consider the impact of nanoparticle shape at different size regimes on targeting, cytotoxicity, and cellular mechanics.

Emergence of Nanoplastic in the Environment and Possible Impact on Human Health.

On account of environmental concerns, the fate and adverse effects of plastics have attracted considerable interest in the past few years. Recent studies have indicated the potential for fragmentation of plastic materials into nanoparticles, i.e., "nanoplastics," and their possible accumulation in the environment. Nanoparticles can show markedly different chemical and physical properties than their bulk material form. Therefore possible risks and hazards to the environment need to be considered and addressed. However, the fate and effect of nanoplastics in the (aquatic) environment has so far been little explored. In this review, we aim to provide an overview of the literature on this emerging topic, with an emphasis on the reported impacts of nanoplastics on human health, including the challenges involved in detecting plastics in a biological environment. We first discuss the possible sources of nanoplastics and their fates and effects in the environment and then describe the possible entry routes of these particles into the human body, as well as their uptake mechanisms at the cellular level. Since the potential risks of environmental nanoplastics to humans have not yet been extensively studied, we focus on studies demonstrating cell responses induced by polystyrene nanoparticles. In particular, the influence of particle size and surface chemistry are discussed, in order to understand the possible risks of nanoplastics for humans and provide recommendations for future studies.

Assessment of the potential for in-plume sulphur dioxide gas-ash interactions to influence the respiratory toxicity of volcanic ash.

BACKGROUND: Volcanic plumes are complex environments composed of gases and ash particles, where chemical and physical processes occur at different temperature and compositional regimes. Commonly, soluble sulphate- and chloride-bearing salts are formed on ash as gases interact with ash surfaces. Exposure to respirable volcanic ash following an eruption is potentially a significant health concern. The impact of such gas-ash interactions on ash toxicity is wholly un-investigated. Here, we study, for the first time, whether the interaction of volcanic particles with sulphur dioxide (SO2) gas, and the resulting presence of sulphate salt deposits on particle surfaces, influences toxicity to the respiratory system, using an advanced in vitro approach. METHODS: To emplace surface sulphate salts on particles, via replication of the physicochemical reactions that occur between pristine ash surfaces and volcanic gas, analogue substrates (powdered synthetic volcanic glass and natural pumice) were exposed to SO2 at 500 °C, in a novel Advanced Gas-Ash Reactor, resulting in salt-laden particles. The solubility of surface salt deposits was then assessed by leaching in water and geochemical modelling. A human multicellular lung model was exposed to aerosolised salt-laden and pristine (salt-free) particles, and incubated for 24 h. Cell cultures were subsequently assessed for biological endpoints, including cytotoxicity (lactate dehydrogenase release), oxidative stress (oxidative stress-related gene expression; heme oxygenase 1 and NAD(P)H dehydrogenase [quinone] 1) and its (pro-)inflammatory response (tumour necrosis factor α, interleukin 8 and interleukin 1ß at gene and protein levels). RESULTS: In the lung cell model no significant effects were observed between the pristine and SO2-exposed particles, indicating that the surface salt deposits, and the underlying alterations to the substrate, do not cause acute adverse effects in vitro. Based on the leachate data, the majority of the sulphate salts from the ash surfaces are likely to dissolve in the lungs prior to cellular uptake. CONCLUSIONS: The findings of this study indicate that interaction of volcanic ash with SO2 during ash generation and transport does not significantly affect the respiratory toxicity of volcanic ash in vitro. Therefore, sulphate salts are unlikely a dominant factor controlling variability in in vitro toxicity assessments observed during previous eruption response efforts.