The Effect of Substrate Properties on Cellular Behavior and Nanoparticle Uptake in Human Fibroblasts and Epithelial Cells.

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.

Cellular Uptake of Silica Particles Influences EGFR Signaling Pathway and is Affected in Response to EGF.

Background: 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.

Substrate stiffness reduces particle uptake by epithelial cells and macrophages in a size-dependent manner through mechanoregulation.

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.

Multi-Functionalized Heteroduplex Antisense Oligonucleotides for Targeted Intracellular Delivery and Gene Silencing in HeLa Cells.

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.

Intracellular gold nanoparticles influence light scattering and facilitate amplified spontaneous emission generation.

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 Uptake of Silica and Gold Nanoparticles Induces Early Activation of Nuclear Receptor NR4A1.

The approval of new nanomedicines requires a deeper understanding of the interaction between cells and nanoparticles (NPs). Silica (SiO2) and gold (Au) NPs have shown great potential in biomedical applications, such as the delivery of therapeutic agents, diagnostics, and biosensors. NP-cell interaction and internalization can trigger several cellular responses, including gene expression regulation. The identification of differentially expressed genes in response to NP uptake contributes to a better understanding of the cellular processes involved, including potential side effects. We investigated gene regulation in human macrophages and lung epithelial cells after acute exposure to spherical 60 nm SiO2 NPs. SiO2 NPs uptake did not considerably affect gene expression in epithelial cells, whereas five genes were up-regulated in macrophages. These genes are principally related to inflammation, chemotaxis, and cell adhesion. Nuclear receptor NR4A1, an important modulator of inflammation in macrophages, was found to be up-regulated. The expression of this gene was also increased upon 1 h of macrophage exposure to spherical 50 nm AuNPs and 200 nm spherical SiO2 NPs. NR4A1 can thus be an important immediate regulator of inflammation provoked by NP uptake in macrophages.

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.

An Inflamed Human Alveolar Model for Testing the Efficiency of Anti-inflammatory Drugs in vitro.

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-1ß) 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.