A novel In Vitro Model for Studying Nanoparticle Interactions with the Small Intestine

Manufactured nanomaterials provide promising features for new technologies in cosmetic, food, and pharmaceutical applications. On the other hand, orally ingested nanomaterials/ nanoparticles may interact with or enter intestinal cells via different mechanisms, resulting in possible injuries of the biological system. For that reason, the current study aims to provide useful information concerning physicochemical properties of nanoparticles with regard to cytotoxic effects and uptake mechanisms in the small intestine. Differently charged polystyrene nanoparticles were used and cytotoxicity and uptake were studied with an intestinal in vitro co-culture model, mimicking the villus epithelium and a triple-culture model recapitulating the follicle-associated epithelium. Mechanisms of cellular transport were investigated at 37°C and 4°C to verify that internalization mainly occurs energy-dependently. Chemical inhibitors (i.e., chlorpromazine, genistein, dynasore) were used to block dynamin-dependent endocytic pathways without affecting cell viability and membrane integrity. Qualification and quantification were performed via confocal microscopy and flow cytometry. Furthermore, co-localization studies with commonly used markers (i.e., transferrin, lactosylceramide) were carried out and co-localization was assessed via calculation of Pearson´s correlation coefficient and Mander´s overlap coefficient. The results show that size and surface chemistry play a crucial role in cytotoxic interactions and cellular uptake of nanoparticles (NPs). Independent of the surface charge, NPs strongly interact with intestinal mucus and are immobilized. Uptake predominantly occurs via M cells and is surface-charge dependent. Whereas negatively charged particles fail to enter cells, positive and neutral particles penetrate M cells energy-dependently. More precisely, both clathrin- and caveolae-mediated endocytosis are involved. It can be concluded that the presented system serves as a valuable tool to assess safety aspects of manufactured nanomaterials and hence, substantially contributes to nanosafety efforts.

Cerium dioxide nanoparticles selectively up-regulate C-C chemokine receptor 2 and CD16 expression on human monocytes

Cerium dioxide nanoparticles (CeO2 NPs) are known as scavengers of reactive oxygen species for the coexistence of Ce3+/ Ce4+ oxidation states. Cell treatments with CeO2 NPs often lead to controversial pro-inflammatory and anti-inflammatory results. The aim of the study was to investigate the immune events following the administration of ceria nanoparticles to THP-1 monocytes. To address this issue, we performed flow cytometry, chemotaxis and ELISA experiments on THP-1 monocytes treated with different concentrations of CeO2 NPs. CeO2 nanoparticle treatments induced a significant pro-inflammatory C-C chemokine receptor 2 (CCR2) up-regulation within the first 6 hours lasting over-expressed for 24 hours. Differently, CCR5 showed no response at any concentration tested. Enhanced chemotaxis towards the CCR2 specific ligand MCP-1 reinforced the observation demonstrating a functional immune outcome. The pro-inflammatory profile of the treated monocytes was also supported by CD16 up-regulation but no differences in CX3CR1 or other monocyte receptors, like CD11b and CD14, were detectable. Moreover, CeO2 NPs exposure did not promote any release of inflammatory cytokines suggesting a specific and direct effect of the nanoparticles on CCR2 and CD16. Our in vitro results reveal a specific role of CeO2 NPs in the up-regulation of CCR2, which might contribute to increase the pro-inflammatory monocyte/macrophage migration toward the sites of CCL2 expression.

Human epithelial cells in vitro – Are they an advantageous tool to help understand the nanomaterial-biological barrier interaction?

AbstratThe human body can be exposed to nanomaterials through a variety of different routes. As nanomaterials get in contact with the skin, the gastrointestinal tract, and the respiratory tract, these biological compartments are acting as barriers to the passage of nano-sized materials into the organism. These structural and functional barriers are provided by the epithelia serving as an interface between biological compartments. In order to initiate the reduction, refinement and replacement of time consuming, expensive and stressful (to the animals) in vivo experimental approaches, many in vitro epithelial cell culture models have been developed during the last decades. This review therefore, focuses on the functional as well as structural aspects of epithelial cells as well as the most commonly used in vitro epithelial models of the primary biological barriers with which nanomaterials might come in contact with either occupationally, or during their manufacturing and application. The advantages and disadvantages of the different in vitro models are discussed in order to provide a clear overview as to whether or not epithelial cell cultures are an advantageous model to be used for basic mechanism and nanotoxicology research.

Transport of Metal Oxide Nanoparticles Across Calu-3 Cell Monolayers Modelling the Air-Blood Barrier

As inhalation is the major exposure route for nanoparticles, the question if inhaled particles can overcome the respiratory epithelial barrier and hence enter the body is of great interest. Here, we adapted the for soluble substances well established Calu-3 in vitro air-blood barrier model to the use of nanoparticle transport testing. As the usually used filter supports hindered particle transport due to their small pore size, supports with a pore size of 3 μm had to be used. On those filters, barrier and transport characteristics of the cells were tested and culture conditions changed to obtain optimal conditions. Functionality was confirmed with transport experiments with polystyrene model particles prior to testing of industrially relevant engineered metal oxide particles. Except for CeO2 nanoparticles, no transport across the epithelial barrier model could be detected. Paracellular permeability and barrier function was not affected by any of the nanoparticles, except for ZrO2.

Important issues in the cytotoxicity screening of nano-sized materials

Due to their extraordinary properties nano-sized materials (NMs) are increasingly used in industrial, pharmaceutical and medical applications. An even broader use is currently limited by concern about their potential adverse effect on health. Screening for toxic effects of all engineered NMs therefore, is needed to demonstrate biocompatibility. The identification of adverse cellular effects is one of the first steps in the toxicological assessment of drug compounds before they get to the market. A panel of cytotoxicity screening assays is available and can be used also for the assessment of NMs. The use of these established and validated assays for the testing of NMs, however, is complicated by the fact that NMs may interfere by color, chemical reactivity and light scattering leading to false positive or false negative results. The paper illustrates the principles of conventional cytotoxicity screening assays and discusses their suitability for the assessment of NMs. Adequate controls to identify interference and alternatives, if interference with the used assay is seen, are suggested.

Health Risks of Nanotechnology

Nanotechnology is one of the key technologies of the 21st century and is associated with high expectations. Products with completely new properties for application in medicine, science, industry and various techniques are designed. However, the larger surface area of nanoparticles makes them highly reactive compared to larger sized particles of the same chemistry resulting in both, desirable and undesirable effects. The need for toxicological data has become increasingly important, thus several international projects are ongoing throughout the European Union. The question concerning the risks for the health and environment should not be disregarded.