Quantification of internalised silica nanoparticles via STED microscopy
In the field of nanosafety, dosimetry is one important parameter for the understanding of nanoparticle (NP) induced effects on a cellular scale as well as for risk assessment. Not only in vivo but also for in vitro experiments, it is critical to have information about the administered NP dose initially added, the delivered dose, comprising the particles reaching the cells via diffusion and sedimentation, and the intracellular dose, the NPs that are internalised by the cells.
In this study, the internalisation of 25 nm and 85 nm silica NPs in A549 cells, a model for type II alveolar epithelial cells, was quantified. Cells were exposed to equal initial NP number concentrations (9.2×10^10 NPs ml^-1) of each particle size. Due to sedimentation, the NP number concentration immediately surrounding the cells was 2.3×10^12 NPs ml^-1.
The number of internalised NPs was extracted from 3D super-resolution STED (stimulated emission depletion) image stacks of entire cells via image processing. Taking the formation of small agglomerates into account that could not be resolved by STED in case of the 25 nm NPs, the number of internalised NPs was estimated to be in the same range (2.5 10^11-4.8 10^12 NPs ml^-1 cell volume) at both particle sizes, with only a slightly stronger accumulation of the 25 nm NPs. The intracellular NP concentration did not significantly exceed the delivered NP concentration within 5 h. The experiments were performed at non-cytotoxic NP concentrations, excluding an influence of cytotoxicity on NP uptake.
Henrike Peuschel, Thomas Ruckelshausen, Christian Cavelius, and Annette Kraegeloh “Quantification of internalized silica nanoparticles via STED microscopy”, BioMed Research International, Article ID 961208.
Senescence and organ ageing induced by carbon nanoparticles
The groups of Prof Judith Haendeler and Dr Klaus Unfried at IUF demonstrated for the first time that pure carbon nanoparticles induce features of cellular senescence in primary endothelial cells and lung epithelial cells (Büchner et al., 2013). This exposure also increases reactive oxygen species (ROS), which are believed to be involved in ageing and age-associated diseases. During the ageing process, a functional decline of organs occurs, resulting in multiple organ failure and death. One hallmark of ageing is the occurrence of cellular senescence. Several studies demonstrated that senescent cells are present in vivo and are less functional than non-senescent cells. Now, the molecular mechanisms of carbon nanoparticle-induced cellular senescence will be investigated ex vivo in primary human lung epithelial cells and endothelial cells. This project has recently been granted by DFG as an individual project shared by both groups. The relevance of the findings will be evaluated in an in vivo model with an emphasis on applying realistic doses.
Mechanisms of toxicity triggered by inhaled carbon nanotubes
Two recent papers put forward by IUF contribute to elucidating molecular mechanisms of nanoparticle cell interactions:
- Researchers led by Dr Catrin Albrecht investigated the effects of multi-walled carbon nanotubes (MWCNT) on lungs with regard to their capacity to modify organic tissue (van Berlo et al., 2014). The researchers describe correlations between the physical parameters of the nanotubes and their ability to cause oxidative stress, inflammatory responses and pulmonary fibrosis in cell cultures and in vivo models.
- A second study looks at the significance of signaling events triggered by carbon nanoparticles interacting with the cellular membrane (Autengruber et al., 2014). These processes can be observed both in vitro in epithelial lung cells and in vivo in animal models as well as in inflammatory cells derived from human blood. Using a newly developed preventive strategy that is being tested for its applicability to humans, this specific interaction between nanoparticles and the cellular membrane could be demonstrated to be relevant for the development of inflammatory responses.
Detection of endotoxins in nanoparticle suspensions
Lipopolysaccharides, also known as endotoxins, are components of the outer membrane of Gram-negative bacteria and are able to provoke immune responses in humans. Therefore, nanomaterials need to be tested for endotoxins before their medical application. Concerning studies on the safe application of nanomaterials, endotoxin contamination can be responsible for flawed results and erroneous conclusions. That is why the possible interference of nanoparticles with standard test systems poses a challenge for the generation of reliable results.
Within the scope of the BMBF-funded project “NanoKon – Systematic evaluation of health effects of nanoscale contrast agents”, the issue of interference in testing scenarios was examined systematically. The report has recently been published in the journal Innate Immunity. The studies were able to produce evidence for the interference of silica and iron-oxide silica nanoparticles with the tested assay, the traditional Limulus amebocyte lysate gel clot assay. Depending on concentration and the particle type, the assay could be observed to be either inhibited or enhanced. Overall, the results indicate that immediate interactions between proteins of the testing reagent and the particle surface cause these interferences. The findings contribute to a better understanding of particle interactions with biological molecules.
Melanie Kucki, Christian Cavelius, and Annette Kraegeloh “Interference of silica nanoparticles with traditional Limulus Amebocyte Lysate gel clot assay” Innate Immunity 2014, 20, (3), 327-336