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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

Cells continuously exert forces on their environment and respond to changes in mechanical forces by altering their behaviour. Many pathologies such as cancer and fibrosis are hallmarked by dysregulation in the extracellular matrix, driving aberrant behaviour through mechanotransduction pathways. We demonstrate that substrate stiffness can be used to regulate cellular endocytosis of particles in a size-dependent fashion. Culture of A549 epithelial cells and J774A.1 macrophages on polystyrene/glass (stiff) and polydimethylsiloxane (soft) substrates indicated that particle uptake is increased up to six times for A549 and two times for macrophages when cells are grown in softer environments. Furthermore, we altered surface characteristics through the attachment of submicron-sized particles as a method to locally engineer substrate stiffness and topography to investigate the biomechanical changes which occurred within adherent epithelial cells,i.e.characterization of A549 cell spreading and focal adhesion maturation. Consequently, decreasing substrate rigidity and particle-based topography led to a reduction of focal adhesion size. Moreover, expression levels of Yes-associated protein were found to correlate with the degree of particle endocytosis. A thorough appreciation of the mechanical cues may lead to improved solutions to optimize nanomedicine approaches for treatment of cancer and other diseases with abnormal mechanosignalling.

Oligonucleotide therapeutics, antisense oligonucleotides (ASOs) and short interfering RNA (siRNA) are short synthetic nucleic acid molecules with a promising potential to treat a wide range of diseases. Despite considerable progress in the field, the development of safe and effective delivery systems that target organs and tissues other than the liver is challenging. While keeping possible off-target oligonucleotide interactions and toxicity related to chemical modifications in mind,innovative solutions for targeted intracellular delivery are highly needed. Herein, we report on the design, synthesis and testing of a novel multi-modified and multi-functionalized heteroduplex oligonucleotide (HDO) with respect to its intracellular delivery and its ability to silence genes in HeLa cells. Simultaneously, folic acid- and peptide- labeled HDO show proficient silencing of the green fluorescent protein (GFP) gene with an 84% reduction in the GFP fluorescence. In addition,the Bcl2 HDO achieved effective Bcl2 gene knockdown in the cells. The data show the proficiency of the multi-functionalization strategy and provide an example for advancing the design of safe and efficient forthcoming oligonucleotide therapeutics, such as HDO.

Oligonucleotide therapeutics, antisense oligonucleotides (ASOs) and short interfering RNA (siRNA) are short synthetic nucleic acid molecules with a promising potential to treat a wide range of diseases. Despite considerable progress in the field, the development of safe and effective delivery systems that target organs and tissues other than the liver is challenging. While keeping possible off-target oligonucleotide interactions and toxicity related to chemical modifications in mind, innovative solutions for targeted intracellular delivery are highly needed. Herein, we report on the design, synthesis and testing of a novel multi-modified and multi-functionalized heteroduplex oligonucleotide (HDO) with respect to its intracellular delivery and its ability to silence genes in HeLa cells. Simultaneously, folic acid- and peptide- labeled HDO show proficient silencing of the green fluorescent protein (GFP) gene with an 84% reduction in the GFP fluorescence. In addition, the Bcl2 HDO achieved effective Bcl2 gene knockdown in the cells. The data show the proficiency of the multi-functionalization strategy and provide an example for advancing the design of safe and efficient forthcoming oligonucleotide therapeutics, such as HDO.

Toxicology assessment using human lung tissue models is critical and complementary for determining the hazards for aerosolized materials. In the present study, we investigated the functional, morphological and reactivity features of reconstituted human airway epithelium (MucilAir™) derived with cells from healthy and asthmatic donors. Tissues were combined with primary human monocyte derived macrophages (MDMs), and response to single Dörentrup Quartz (DQ12) dosage (10 μg/cm2) exposure over 10 days was investigated. Our study showed that DQ12 in healthy and asthmatic tissues with or without MDMs did not affect cell viability and tight junctions’ formations for up to 10 days. Pre-stained macrophages seeded on top of the healthy tissues moved towards the insert's edges, whereas a more constrained movement was observed for macrophages seeded on top of the asthmatic tissues. The reduced macrophage motility in asthmatic tissues can be attributed to stronger mucus production under pathophysiological, i.e., asthmatic, conditions and related restriction of the cilia beating effect. In addition, we found that in healthy tissues, the overexpression of transforming growth factor (TGF)-β and interleukin (IL)-8 was more pronounced following exposure to DQ12, but the addition of MDMs mitigated the inflammatory reaction of DQ12. Our findings emphasize the potential key role of macrophages added to the 3D human airway epithelia model in orchestrating the significant inflammatory responses generated by DQ12 exposure.

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., This review contributes to the current understanding of NPs cellular uptake and gives an overview about molecules, which can enhance or decrease cellular internalization of NPs.

Generation of amplified stimulated emission inside mammalian cells has paved the way for a novel bioimaging and cell sensing approach. Single cells carrying gain media (e.g., fluorescent molecules) are placed inside an optical cavity, allowing the production of intracellular laser emission upon sufficient optical pumping. Here, we investigate the possibility to trigger another amplified emission phenomenon (i.e., amplified spontaneous emission or ASE) inside two different cell types, namely macrophage and epithelial cells from different species and tissues, in the presence of a poorly reflecting cavity. Furthermore, the resulting ASE properties can be enhanced by introducing plasmonic nanoparticles. The presence of gold nanoparticles (AuNPs) in rhodamine 6G-labeled A549 epithelial cells results in higher intensity and lowered ASE threshold in comparison to cells without nanoparticles, due to the effect of plasmonic field enhancement. An increase in intracellular concentration of AuNPs in rhodamine 6G-labeled macrophages is, however, responsible for the twofold increase in the ASE threshold and a reduction in the ASE intensity, dominantly due to a suppressed in and out-coupling of light at high nanoparticle concentrations.

Cellular surface recognition and behavior are driven by a host of physical and chemical features which have been exploited to influence particle-cell interactions. Mechanical and topographical cues define the physical milieu which plays an important role in defining a range of cellular activities such as material recognition, adhesion, and migration through cytoskeletal organization and signaling. In order to elucidate the effect of local mechanical and topographical features generated by the adsorption of particles to an underlying surface on primary human monocyte-derived macrophages (MDM), a series of poly(N-isopropylacrylamide) (pNIPAM) particles with differing rigidity are self-assembled to form a defined particle-decorated surface. Assembly of particle-decorated surfaces is facilitated by modification of the underlying glass to possess a positive charge through functionalization using 3-aminopropyltriethoxysilane (APTES) or coating with poly(L-lysine) (PLL). MDMs are noted to preferentially remove particles with higher degrees of crosslinking (stiffer) than those with lower degrees of crosslinking (softer). Alterations to the surface density of particles enabled a greater area of the particle-decorated surface to be cleared. Uniquely, the impact of particle adsorption is evinced to have a direct impact on topographical recognition of the surface, suggesting a novel approach for controllably affecting cell-surface recognition and response.