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Jana Fleddermann,1 Julia Susewind,2 Henrike Peuschel,1 Marcus Koch,1 Isabella Tavernaro,1 Annette Kraegeloh1 1INM – Leibniz Institute for New Materials, Saarbrücken, Germany; 2Pharmacelsus GmbH, Saarbrücken, Germany Introduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials.Materials and methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy.Results: Spheroids were exposed to 100 µg mL-1 SiO2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO2 NPs during the process of spheroid formation had no impact on final spheroid size.Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation. Keywords: silica nanoparticles, human hepatocarcinoma cells, spheroids, penetration

Abstract Background Particulate air pollution in lung epithelial cells induces pathogenic endpoints like proliferation, apoptosis, and pro-inflammatory reactions. The activation of the epidermal growth factor receptor (EGFR) is a key event responsible for signalling events involving mitogen activated protein kinases specific for these endpoints. The molecular events leading to receptor activation however are not well understood. These events are relevant for the toxicological evaluation of inhalable particles as well as for potential preventive strategies in situations when particulate air pollution cannot be avoided. The current study therefore had the objective to elucidate membrane-coupled events leading to EGFR activation and the subsequent signalling cascade in lung epithelial cells. Furthermore, we aimed to identify the molecular target of ectoine, a biophysical active substance which we described to prevent carbon nanoparticle-induced lung inflammation. Methods Membrane signalling events were investigated in isolated lipid rafts from lung epithelial cells with regard to lipid and protein content of the signalling platforms. Using positive and negative intervention approaches, lipid raft changes, subsequent signalling events, and lung inflammation were investigated in vitro in lung epithelial cells (RLE-6TN) and in vivo in exposed animals. Results Carbon nanoparticle treatment specifically led to an accumulation of ceramides in lipid rafts. Detailed analyses demonstrated a causal link of ceramides and subsequent EGFR activation coupled with a loss of the receptor in the lipid raft fractions. In vitro and in vivo investigations demonstrate the relevance of these events for carbon nanoparticle-induced lung inflammation. Moreover, the compatible solute ectoine was able to prevent ceramide-mediated EGFR phosphorylation and subsequent signalling as well as lung inflammation in vivo. Conclusion The data identify a so far unknown event in pro-inflammatory signalling and contribute to the understanding of particle cell interaction and therefore to risk identification and risk assessment of inhalable xenobiotics. Moreover, as this cellular reaction can be prevented by the well tolerated substance ectoine, a molecular preventive strategy for susceptible persons against airway inflammation is proposed.

The detection of 2-chloroethanol in foods generally follows an assumption that the pesticide ethylene oxide has been used at some stage in the supply chain. In this situation the Pesticide Residues in Food Regulation (EC) 396/2005 requires 2-chloroethanol to be assessed as if equivalent to ethylene oxide, which has been classified as a genotoxic carcinogen. This review investigated whether this is an appropriate risk assessment approach for 2-chloroethanol. This involved an assessment of existing genotoxicity and carcinogenicity data, application of Structure Activity Based Read Across for carcinogenicity assessment, biological reactivity in the ToxTracker assay and micronuclei formation in HepaRG cells. Although we identified there is an absence of a standard oral bioassay for 2-chloroethanol, carcinogenicity weight-of-evidence assessment along with data on relevant structural analogues do not show evidence for carcinogenicity for 2-chloroethanol. The absence of genotoxicity was demonstrated for 2-chloroethanol and suitable analogues. In contrast, ethylene oxide showed reactivity towards markers indicative of direct DNA damage which is consistent with what is known about its mode-of-action. These data facilitate the understanding of 2-chloroethanol and given that it is not a genotoxic carcinogen suggest it must be assessed relative to non-cancer endpoints and a health protective Reference Dose should be established on that basis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ashley Allemang, Stefan Pfuhler, Cathy Lester, Kirstin Kosemund, Catherine Mahony and Lara O’ Keeffe are all employees of a company that includes manufacture of food supplements.Thomas Roth and Henrike Peuschel are employees at SCC GmbH in Germany and were paid by Kappa Biosciences AS to prepare the ToxTracker assay part of the initial manuscript. Trygve Bergeland is a shareholder and employee of Kappa Bioscience AS., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: Nanoparticles (NPs) are used in numerous products in technical fields and biomedicine; their potential adverse effects have to be considered in order to achieve safe applications. Besides their distribution in tissues, organs, and cellular localization, their impact and penetration during the process of tissue formation occurring in vivo during liver regeneration are critical steps for establishment of safe nanomaterials., Materials and Methods: In this study, 3D cell culture of human hepatocarcinoma cells (HepG2) was used to generate cellular spheroids, serving as in vitro liver microtissues. In order to determine their differential distribution and penetration depth in HepG2 spheroids, SiO 2 NPs were applied either during or after spheroid formation. The NP penetration was comprehensively studied using confocal laser scanning microscopy and scanning electron microscopy., Results: Spheroids were exposed to 100 µg mL -1 SiO 2 NPs either at the beginning of spheroid formation, or during or after formation of spheroids. Microscopy analyses revealed that NP penetration into the spheroid is limited. During and after spheroid formation, SiO 2 NPs penetrated about 20 µm into the spheroids, corresponding to about three cell layers. In contrast, because of the addition of SiO 2 NPs simultaneously to cell seeding, NP agglomerates were located also in the spheroid center. Application of SiO 2 NPs during the process of spheroid formation had no impact on final spheroid size., Conclusion: Understanding the distribution of NPs in tissues is essential for biomedical applications. The obtained results indicate that NPs show only limited penetration into already formed tissue, which is probably caused by the alteration of the tissue structure and cell packing density during the process of spheroid formation., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

In recent years, fluorescent nanomaterials have gained high relevance in biological applications as probes for various fluorescence-based spectroscopy and imaging techniques. Among these materials, dye-doped silica nanoparticles have demonstrated a high potential to overcome the limitations presented by conventional organic dyes such as high photobleaching, low stability and limited fluorescence intensity. In the present work we describe an effective approach for the preparation of fluorescent silica nanoparticles in the size range between 15 and 80 nm based on L-arginine-controlled hydrolysis of tetraethoxysilane in a biphasic cyclohexane-water system. Commercially available far-red fluorescent dyes (Atto647N, Abberior STAR 635, Dy-647, Dy-648 and Dy-649) were embedded covalently into the particle matrix, which was achieved by aminosilane coupling. The physical particle attributes (particle size, dispersion, degree of agglomeration and stability) and the fluorescence properties of the obtained particles were compared to particles from commonly known synthesis methods. As a result, the spectroscopic characteristics of the presented monodisperse dye-doped silica nanoparticles were similar to those of the free uncoupled dyes, but indicate a much higher photostability and brightness. As revealed by dynamic light scattering and ζ-potential measurements, all particle suspensions were stable in water and cell culture medium. In addition, uptake studies on A549 cells were performed, using confocal and stimulated emission depletion (STED) microscopy. Our approach allows for a step-by-step formation of dye-doped silica nanoparticles in the form of dye-incorporated spheres, which can be used as versatile fluorescent probes in confocal and STED imaging.

Background: Quantum dots (QDs) have great potential as fluorescent labels but cytotoxicity relating to extra- and intracellular degradation in biological systems has to be addressed prior to biomedical applications. In this study, human intestinal cells (Caco-2) grown on transwell membranes were used to study penetration depth, intracellular localization, translocation and cytotoxicity of CdSe/ZnS QDs with amino and carboxyl surface modifications. The focus of this study was to compare the penetration depth of QDs in differentiated vs undifferentiated cells using confocal microscopy and image processing., Results: Caco-2 cells were exposed to QDs with amino (NH 2 ) and carboxyl (COOH) surface groups for 3 days using a concentration of 45 µg cadmium ml -1 . Image analysis of confocal/multiphoton microscopy z-stacks revealed no penetration of QDs into the cell lumen of differentiated Caco-2 cells. Interestingly, translocation of cadmium ions onto the basolateral side of differentiated monolayers was observed using high resolution inductively coupled plasma mass spectrometry (ICP-MS). Membrane damage was neither detected after short nor long term incubation in Caco-2 cells. On the other hand, intracellular localization of QDs after exposure to undifferentiated cells was observed and QDs were partially located within lysosomes., Conclusions: In differentiated Caco-2 monolayers, representing a model for small intestinal enterocytes, no penetration of amino and carboxyl functionalized CdSe/ZnS QDs into the cell lumen was detected using microscopy analysis and image processing. In contrast, translocation of cadmium ions onto the basolateral side could be detected using ICP-MS. However, even after long term incubation, the integrity of the cell monolayer was not impaired and no cytotoxic effects could be detected. In undifferentiated Caco-2 cells, both QD modifications could be found in the cell lumen. Only to some extend, QDs were localized in endosomes or lysosomes in these cells. The results indicate that the differentiation status of Caco-2 cells is an important factor in internalization and localization studies using Caco-2 cells. Furthermore, a combination of microscopy analysis and sensitive detection techniques like ICP-MS are necessary for studying the interaction of cadmium containing QDs with cells.

In this study, we report p-coumaric acid as novel and effective response marker for indirectly measuring the levels of hypoxia in normal primary bronchial epithelial cells. We developed a simple and rapid technique based on hydrophilic interaction chromatography-electrospray ionization-mass spectrometry (HILIC-ESI-MS). During 168h of hypoxia without induction of reactive oxygen species (ROS), an almost linear increase of p-coumaric acid levels was observed. We interpret the increasing p-coumaric acid concentrations during hypoxia as a result of cell damage, triggered by reduced co-enzyme Q10 levels, because the oxidative cascade was not able to supply sufficient energy. The HILIC-ESI-MS assay within p-coumaric acid exhibited a linear dynamic range from 60 to 610 ng/μL with correlation coefficient of 0.9998. The precision of the assay was ≤15% RSD and method accuracies between 97 and 108%., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The development of safe engineered nanoparticles (NPs) requires a detailed understanding of their interaction mechanisms on a cellular level. Therefore, quantification of NP internalization is crucial to predict the potential impact of intracellular NP doses, providing essential information for risk assessment as well as for drug delivery applications. In this study, the internalization of 25 nm and 85 nm silica nanoparticles (SNPs) in alveolar type II cells (A549) was quantified by application of super-resolution STED (stimulated emission depletion) microscopy. Cells were exposed to equal particle number concentrations (9.2 × 10(10) particles mL(-1)) of each particle size and the sedimentation of particles during exposure was taken into account. Microscopy images revealed that particles of both sizes entered the cells after 5 h incubation in serum supplemented and serum-free medium. According to the in vitro sedimentation, diffusion, and dosimetry (ISDD) model 20-27% of the particles sedimented. In comparison, 10(2)-10(3) NPs per cell were detected intracellularly serum-containing medium. Furthermore, in the presence of serum, no cytotoxicity was induced by the SNPs. In serum-free medium, large agglomerates of both particle sizes covered the cells whereas only high concentrations (≥ 3.8 × 10(12) particles mL(-1)) of the smaller particles induced cytotoxicity.

Nanoparticles may be taken up into cells via endocytotic processes whereby the foreign particles are encapsulated in vesicles formed by lipid bilayers. After uptake into these endocytic vesicles, intracellular targeting processes and vesicle fusion might cause transfer of the vesicle cargo into other vesicle types, e.g., early or late endosomes, lysosomes, or others. In addition, nanoparticles might be taken up as single particles or larger agglomerates and the agglomeration state of the particles might change during vesicle processing. In this study, liposomes are regarded as simple models for intracellular vesicles. We compared the energetic balance between two liposomes encapsulating each a single silica nanoparticle and a large liposome containing two silica nanoparticles. Analytical expressions were derived that show how the energy of the system depends on the particle size and the distance between the particles. We found that the electrostatic contributions to the total energy of the system are negligibly small. In contrast, the van der Waals term strongly favors arrangements where the liposome snugly fits around the nanoparticle(s). Thus the two separated small liposomes have a more favorable energy than a larger liposome encapsulating two nanoparticles.

The life span of neutrophilic granulocytes has a determining impact on the intensity and duration of neutrophil driven lung inflammation. Based on the compatible solute ectoine, we aimed to prevent anti-apoptotic reactions in neutrophils triggered by the inflammatory microenvironment in the lung. Neutrophils from chronic obstructive pulmonary disease patients and control individuals were exposed to inflammatory mediators and xenobiotics in the presence or absence of ectoine. The in vivo relevance of this approach was tested in xenobiotic-induced lung inflammation in rats. The reduction of apoptosis rates of ex vivo-exposed neutrophils from all study groups was significantly restored in the presence of ectoine. However, natural apoptosis rates not altered by inflammatory stimuli were not changed by ectoine. Mechanistic analyses demonstrated the preventive effect of ectoine on the induction of anti-apoptotic signalling. Neutrophilic lung inflammation induced by single or multiple expositions of animals to environmental particles was reduced after the therapeutic intervention with ectoine. Analyses of neutrophils from bronchoalveolar lavage indicate that the in vivo effect is due to the restoration of neutrophil apoptosis. Ectoine, a compound of the highly compliant group of compatible solutes, demonstrates a reproducible and robust effect on the resolution of lung inflammation.

Owing to their specific physico/chemical properties, engineered as well as environmental nanoparticles can induce pathogenic endpoints in humans. Earlier studies demonstrated that pure carbon nanoparticles induce cell signaling events at the level of membrane receptor activation in lung epithelial cells. As a possible link between receptor activation and subsequent MAP-kinase signaling, the involvement of Src family kinases was investigated in cell lines of organs potentially exposed to environmental nanoparticles. Human cells from bronchus, intestine, and skin (keratinocytes) as well as rat lung epithelial cells showed similar time patterns for the activation of mitogen-activated protein kinases Erk1/2 as well as Src family kinases (SFK) when treated with carbon nanoparticles. Moreover, c-Src was identified as an integral part of the signaling mediating the transfer of information from membrane receptors to members of the proliferative signaling cascade in lung epithelial cells. Pretreatment of cells with the compatible solute ectoine, which is known to stabilize macromolecules, reduced the nanoparticle specific phosphorylation of SFK. Together with earlier in vivo and in vitro data, this demonstrates that compatible solutes prevent nanoparticle-induced signaling steps at the level of membrane-coupled signaling.

Cell-membrane-dependent proliferative signal transduction activated by ultrafine carbon particles in lung epithelial cells involves the specific induction of Akt and ERK1/2 phosphorylation. Particle-induced generation of reactive oxygen species (ROS) and oxidative stress are regarded as initial molecular mechanisms leading to the induction of diverse cellular responses. Therefore, we aimed to analyze the ROS dependence of the induced activation of the Akt/ERK1/2 signaling pathway upon exposure to ultrafine particulate matter (UPM). For this, ultrafine carbon black (ufCB) and ferric sulfate (FS) were used as a model representing the carbonaceous core and a nonparticulate Fenton-reactive transition metal salt often found in combustion-derived UPM. Cell-free as well as intracellular particle-induced ROS generation was assessed and related to the induced Akt and ERK1/2 phosphorylation by inhibiting oxidative stress with catalase, superoxide dismutase, and N-acetylcysteine. We show here that the activation of this signal transduction pathway was mainly due to intracellular, rather than extracellular, ROS production induced by both ufCB and FS. Further inhibitor studies on the role of cell membrane receptors pointed to the epidermal growth factor receptor as a common mediator for particle- as well as transition metal-induced signaling, whereas integrin-dependent Akt and ERK1/2 activation seems to be particle-specific., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)