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Mauro Sousa de Almeida,1 Arya Roshanfekr,1 Sandor Balog,1 Alke Petri-Fink,1,2 Barbara Rothen-Rutishauser1 1Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland; 2Department of Chemistry, University of Fribourg, Fribourg, SwitzerlandCorrespondence: Barbara Rothen-Rutishauser, Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland, Email barbara.rothen@unifr.chBackground: The human epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is involved in several key cellular processes, such as cell proliferation and differentiation, and it has been linked to the development and progression of various cancers (e.g., breast and lung). Researchers have attempted to improve current cancer-targeted therapies by conjugating molecules on the surface of (nano)particles to efficiently target and inhibit EGFR. However, very few in vitro studies have investigated the effect of particles per se on EGFR signaling and dynamics. Furthermore, the impact of concomitant exposure of particles and EGFR ligands, such as epidermal growth factor (EGF) on cellular uptake efficiency has received little attention.Purpose: The purpose of this research was to determine the effects of silica (SiO2) particles on EGFR expression and intracellular signaling pathways in A549 lung epithelial cells, in the presence or absence of epidermal growth factor (EGF).Results: We showed that A549 cells are able to internalize SiO2 particles with core diameters of 130 nm and 1 μm without affecting cell proliferation or migration. However, both SiO2 particles interfere with the EGFR signaling pathway by raising the endogenous levels of extracellular signal-regulated kinase (ERK) 1/2. Furthermore, both in the presence and absence of SiO2 particles, the addition of EGF increased cell migration. EGF also stimulated cellular uptake of 130 nm SiO2 particles but not 1 μm particles. The increased uptake is primarily associated with EGF-stimulated macropinocytosis.Conclusion: This study shows that SiO2 particle uptake interferes with cellular signaling pathways and can be boosted by concurrent exposure to the bioactive molecule EGF. SiO2 particles, both alone and in combination with the ligand EGF, interfere with EGFR signaling pathway in a size-dependent manner.Keywords: endocytosis, nanoparticles, epidermal growth factor, cellular signaling

Emilie Seydoux,1,2 Barbara Rothen-Rutishauser,1,3 Izabela M Nita,1 Sandor Balog,3 Amiq Gazdhar,1 Philip A Stumbles,4,5 Alke Petri-Fink,3,6 Fabian Blank,1,* Christophe von Garnier1,*1Department of Respiratory Medicine, Inselspital, Bern University Hospital, Department of Clinical Research, University of Bern, 2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland; 3Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland; 4School of Veterinary and Life Sciences, Molecular and Biomedical Sciences, Murdoch University, Perth, WA, Australia; 5Telethon Kids Institute, Perth, WA, Australia; 6Department of Chemistry, University of Fribourg, Fribourg, Switzerland*These authors contributed equally to the manuscriptIntroduction: Nanosized particles may enable therapeutic modulation of immune responses by targeting dendritic cell (DC) networks in accessible organs such as the lung. To date, however, the effects of nanoparticles on DC function and downstream immune responses remain poorly understood. Methods: Bone marrow–derived DCs (BMDCs) were exposed in vitro to 20 or 1,000 nm polystyrene (PS) particles. Particle uptake kinetics, cell surface marker expression, soluble protein antigen uptake and degradation, as well as in vitro CD4+ T-cell proliferation and cytokine production were analyzed by flow cytometry. In addition, co-localization of particles within the lysosomal compartment, lysosomal permeability, and endoplasmic reticulum stress were analyzed. Results: The frequency of PS particle–positive CD11c+/CD11b+ BMDCs reached an early plateau after 20 minutes and was significantly higher for 20 nm than for 1,000 nm PS particles at all time-points analyzed. PS particles did not alter cell viability or modify expression of the surface markers CD11b, CD11c, MHC class II, CD40, and CD86. Although particle exposure did not modulate antigen uptake, 20 nm PS particles decreased the capacity of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4+ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4+ T-cell stimulating capacity, 20 nm (but not 1,000 nm) PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles. Keywords: polystyrene particles, nanoparticles, immune modulation, mouse dendritic cells, CD4+ T-cells

Abstract Despite numerous discussions, workshops, reviews and reports about responsible development of nanotechnology, information describing health and environmental risk of engineered nanoparticles or nanomaterials is severely lacking and thus insufficient for completing rigorous risk assessment on their use. However, since preliminary scientific evaluations indicate that there are reasonable suspicions that activities involving nanomaterials might have damaging effects on human health; the precautionary principle must be applied. Public and private institutions as well as industries have the duty to adopt preventive and protective measures proportionate to the risk intensity and the desired level of protection. In this work, we present a practical, 'user-friendly' procedure for a university-wide safety and health management of nanomaterials, developed as a multi-stakeholder effort (government, accident insurance, researchers and experts for occupational safety and health). The process starts using a schematic decision tree that allows classifying the nano laboratory into three hazard classes similar to a control banding approach (from Nano 3 - highest hazard to Nano1 - lowest hazard). Classifying laboratories into risk classes would require considering actual or potential exposure to the nanomaterial as well as statistical data on health effects of exposure. Due to the fact that these data (as well as exposure limits for each individual material) are not available, risk classes could not be determined. For each hazard level we then provide a list of required risk mitigation measures (technical, organizational and personal). The target 'users' of this safety and health methodology are researchers and safety officers. They can rapidly access the precautionary hazard class of their activities and the corresponding adequate safety and health measures. We succeed in convincing scientist dealing with nano-activities that adequate safety measures and management are promoting innovation and discoveries by ensuring them a safe environment even in the case of very novel products. The proposed measures are not considered as constraints but as a support to their research. This methodology is being implemented at the Ecole Polytechnique de Lausanne in over 100 research labs dealing with nanomaterials. It is our opinion that it would be useful to other research and academia institutions as well.

Abstract 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. Graphical Abstract

Antimony-doped tin oxide nanoparticles (ATO NPs) have emerged as a promising tool in biomedical applications, namely robust photothermal effects upon near-infrared (NIR) light exposure, enabling controlled thermal dynamics to induce spatial cell death. This study investigated the interplay between ATO NPs and macrophages, understanding cellular uptake and cytokine release. ATO NPs demonstrated biocompatibility with no impact on macrophage viability and cytokine secretion. These findings highlight the potential of ATO NPs for inducing targeted cell death in cancer treatments, leveraging their feasibility, unique NIR properties, and safe interactions with immune cells. ATO NPs offer a transformative platform with significant potential for future biomedical applications by combining photothermal capabilities and biocompatibility. [ABSTRACT FROM AUTHOR]

A large number of prevalent lung diseases is associated with tissue inflammation. Clinically, corticosteroid therapies are applied systemically or via inhalation for the treatment of lung inflammation, and a number of novel therapies are being developed that require preclinical testing. In alveoli, macrophages and dendritic cells play a key role in initiating and diminishing pro-inflammatory reactions and, in particular, macrophage plasticity (M1 and M2 phenotypes shifts) has been reported to play a significant role in these reactions. Thus far, no studies with in vitro lung epithelial models have tested the comparison between systemic and direct pulmonary drug delivery. Therefore, the aim of this study was to develop an inflamed human alveolar epithelium model and to test the resolution of LPS-induced inflammation in vitro with a corticosteroid, methylprednisolone (MP). A specific focus of the study was the macrophage phenotype shifts in response to these stimuli. First, human monocyte-derived macrophages were examined for phenotype shifts upon exposure to lipopolysaccharide (LPS), followed by treatment with MP. A multicellular human alveolar model, composed of macrophages, dendritic cells, and epithelial cells, was then employed for the development of inflamed models. The models were used to test the anti-inflammatory potency of MP by monitoring the secretion of pro-inflammatory mediators (interleukin [IL]-8, tumor necrosis factor-α [TNF-α], and IL-1β) through four different approaches, mimicking clinical scenarios of inflammation and treatment. In macrophage monocultures, LPS stimulation shifted the phenotype towards M1, as demonstrated by increased release of IL-8 and TNF-α and altered expression of phenotype-associated surface markers (CD86, CD206). MP treatment of inflamed macrophages reversed the phenotype towards M2. In multicellular models, increased pro-inflammatory reactions after LPS exposure were observed, as demonstrated by protein secretion and gene expression measurements. In all scenarios, among the tested mediators the most pronounced anti-inflammatory effect of MP was observed for IL-8. Our findings demonstrate that our inflamed multicellular human lung model is a promising tool for the evaluation of anti-inflammatory potency of drug candidates in vitro. With the presented setup, our model allows a meaningful comparison of the systemic vs. inhalation administration routes for the evaluation of the efficacy of a drug in vitro.

The delivery of nanomedicines into cells holds enormous therapeutic potential; however little is known regarding how the extracellular matrix (ECM) can influence cell–nanoparticle (NP) interactions. Changes in ECM organization and composition occur in several pathophysiological states, including fibrosis and tumorigenesis, and may contribute to disease progression. We show that the physical characteristics of cellular substrates, that more closely resemble the ECM in vivo, can influence cell behavior and the subsequent uptake of NPs. Electrospinning was used to create two different substrates made of soft polyurethane (PU) with aligned and non-aligned nanofibers to recapitulate the ECM in two different states. To investigate the impact of cell–substrate interaction, A549 lung epithelial cells and MRC-5 lung fibroblasts were cultured on soft PU membranes with different alignments and compared against stiff tissue culture plastic (TCP)/glass. Both cell types could attach and grow on both PU membranes with no signs of cytotoxicity but with increased cytokine release compared with cells on the TCP. The uptake of silica NPs increased more than three-fold in fibroblasts but not in epithelial cells cultured on both membranes. This study demonstrates that cell–matrix interaction is substrate and cell-type dependent and highlights the importance of considering the ECM and tissue mechanical properties when designing NPs for effective cell targeting and treatment. [ABSTRACT FROM AUTHOR]

The gastrointestinal tract is the main target of orally ingested nanoparticles (NPs) and at the same time is exposed to noxious substances, such as bacterial components. We investigated the interaction of 59 nm silica (SiO2) NPs with differentiated Caco-2 intestinal epithelial cells in the presence of cholera toxin subunit B (CTxB) and compared the effects to J774A.1 macrophages. CTxB can affect cellular functions and modulate endocytosis via binding to the monosialoganglioside (GM1) receptor, expressed on both cell lines. After stimulating macrophages with CTxB, we observed notable changes in the membrane structure but not in Caco-2 cells, and no secretion of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) was detected. Cells were then exposed to 59 nm SiO2 NPs and CtxB sequentially and simultaneously, resulting in a high NP uptake in J774A.1 cells, but no uptake in Caco-2 cells was detected. Flow cytometry analysis revealed that the exposure of J774A.1 cells to CTxB resulted in a significant reduction in the uptake of SiO2 NPs. In contrast, the uptake of NPs by highly selective Caco-2 cells remained unaffected following CTxB exposure. Based on colocalization studies, CTxB and NPs might enter cells via shared endocytic pathways, followed by their sorting into different intracellular compartments. Our findings provide new insights into CTxB's function of modulating SiO2 NP uptake in phagocytic but not in differentiated intestine cells. [ABSTRACT FROM AUTHOR]

When nanomaterials enter biological fluids, they are immediately covered by biomolecules, particularly proteins, forming the so-called protein corona. The dynamic nature and complexity of the protein corona can impact upon the biological effects and distribution of nanomaterials with an organism. Therefore, the protein corona is an important factor in determining the biological impact of any nanomaterials. The protein adsorption pattern is determined by various factors, including the bio-fluids’ protein composition, the nanomaterials’ physicochemical properties, as well as the time and type of exposure. Predominantly, research has focused upon spherical nano-objects, however, due to their ever-increasing potential use within human based applications, and, therefore, heightening and inevitable exposure to the human body, little is known regarding how proteins interact with nanofibers. Therefore, the present review focuses on the current knowledge as to how the geometry of man-made (nano)fibers, carbon nanotubes (in comparison with asbestos fibers), affects their interaction with proteins within biological fluids. Summarizing state-of the art methodologies applied to dissect protein-binding signatures, it is further discussed whether the protein corona composition of fibrous and non-fibrous materials differ, as well as what impact the protein corona has on (nano)fiber uptake, intracellular distribution and their subsequent toxicity.