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The breadth of applications of nanoparticles and the access to food-associated consumer products containing nanosized materials lead to oral human exposure to such particles. In biological fluids nanoparticles dynamically interact with biomolecules and form a protein corona. Knowledge about the protein corona is of great interest for understanding the molecular effects of particles as well as their fate inside the human body. We used a mass spectrometry-based toxicoproteomics approach to elucidate mechanisms of toxicity of silver nanoparticles and to comprehensively characterize the protein corona formed around silver nanoparticles in Caco-2 human intestinal epithelial cells. Results were compared with respect to the cellular function of proteins either affected by exposure to nanoparticles or present in the protein corona. A transcriptomic data set was included in the analyses in order to obtain a combined multiomics view of nanoparticle-affected cellular processes. A relationship between corona proteins and the proteomic or transcriptomic responses was revealed, showing that differentially regulated proteins or transcripts were engaged in the same cellular signaling pathways. Protein corona analyses of nanoparticles in cells might therefore help in obtaining information about the molecular consequences of nanoparticle treatment.
The elucidation of mechanisms underlying the cellular uptake of nanoparticles (NPs) is an important topic in nanotoxicological research. Most studies dealing with silver NP uptake provide only qualitative data about internalization efficiency and do not consider NP-specific dosimetry. Therefore, we performed a comprehensive comparison of the cellular uptake of differently coated silver NPs of comparable size in different human intestinal Caco-2 cell-derived models to cover also the influence of the intestinal mucus barrier and uptake-specialized M-cells. We used a combination of the Transwell system, transmission electron microscopy, atomic absorption spectroscopy, and ion beam microscopy techniques. The computational in vitro sedimentation, diffusion, and dosimetry (ISDD) model was used to determine the effective dose of the particles in vitro based on their individual physicochemical characteristics. Data indicate that silver NPs with a similar size and shape show coating-dependent differences in their uptake into Caco-2 cells. The internalization of silver NPs was enhanced in uptake-specialized M-cells while the mucus did not provide a substantial barrier for NP internalization. ISDD modeling revealed a fivefold underestimation of dose–response relationships of NPs in in vitro assays. In summary, the present study provides dosimetry-adjusted quantitative data about the influence of NP coating materials in cellular uptake into human intestinal cells. Underestimation of particle effects in vitro might be prevented by using dosimetry models and by considering cell models with greater proximity to the in vivo situation, such as the M-cell model.
Aluminum has gathered toxicological Attention based on relevant human exposure and its suspected hazardous potential. Nanoparticles from food supplements or Food contact materials may reach the human gastrointestinal tract.
Here, we monitored the physicochemical fate of aluminum containing nanoparticles and aluminum ions when passaging an in vitro model of the human gastrointestinal tract. Smallangle X-ray scattering (SAXS), transmission electron microscopy (TEM), ion beam microscopy (IBM), secondary ion beam mass spectrometry (TOF-SIMS), and inductively coupled plasma mass spectrometry (ICP-MS) in the singleparticle mode were employed to characterize two aluminumcontaining nanomaterials with different particle core materials (Al0, γAl2O3) and soluble AlCl3. Particle size and shape remained unchanged in saliva, whereas strong Agglomeration of both aluminum nanoparticle species was observed at low pH in gastric fluid together with an increased ion release. The levels of free aluminum ions decreased in intestinal fluid and the particles deagglomerated, thus liberating primary particles again. Dissolution of nanoparticles was limited and substantial changes of their shape and size were not detected. The amounts of particle-associated phosphorus, chlorine, potassium, and calcium increased in intestinal fluid, as compared to nanoparticles in standard dispersion.
Interestingly, nanoparticles were found in the intestinal fluid after addition of ionic aluminum. We provide a comprehensive characterization of the fate of aluminum nanoparticles in simulated gastrointestinal fluids, demonstrating that orally ingested nanoparticles probably reach the intestinal epithelium. The balance between dissolution and de novo complex formation should be considered when evaluating nanotoxicological experiments.
Present data indicate that the quantification of silver nanoparticle internalization revealed a clear particle-specific and coatingrelated uptake. Furthermore, a high amount of silver nanoparticles is taken up in cell models of higher complexity. Thus, an underestimation of particle effects in vitro might be prevented by considering cell models with greater proximity to the in vivo situation.
Even although quite a number of studies have been performed so far to demonstrate nanoparticle-specific effects of substances in living systems, clear evidence of these effects is still under debate. The present study was designed as a comparative proteomic analysis of human intestinal cells exposed to a commercial silver nanoparticle reference material and ions from AgNO3. A two-dimensional gel electrophoresis/MALDI mass spectrometry (MS)-based proteomic analysis was conducted after 24-h incubation of differentiated Caco-2 cells with non-cytotoxic and low cytotoxic silver concentrations (2.5 and 25 µg ml−1 nanosilver, 0.5 and 5 µg ml−1 AgNO3). Out of an overall number of 316 protein spots differentially expressed at a fold change of ≥ 1.4 or ≤ −1.4 in all treatments, 169 proteins could be identified. In total, 231 spots were specifically deregulated in particle-treated groups compared with 41 spots, which were limited to AgNO3-treatments. Forty-four spots (14 %) were commonly deregulated by both types of treatment. A considerable fraction of the proteins differentially expressed after treatment with nanoparticles is related to protein folding, synthesis or modification of proteins as well as cellular assembly and organization. Overlays of networks obtained for particulate and ionic treatments showed matches, indicating common mechanisms of combined particle and ionic silver exposure and exclusive ionic silver treatment. However, proteomic responses of Caco-2 cells treated with higher concentrations of silver species also showed some differences, for example regarding proteins related to fatty acid and energy metabolism, suggesting an induction of also some different molecular mechanisms for particle exposure and ionic treatment.
Aluminum (Al) can be taken up from food, packaging, or the environment and thus reaches the human gastrointestinal tract. Its toxic potential after oral uptake is still discussed. The fate of different solid and ionic Al species during the passage through the digestive tract is the focus of this research, as well as the cellular effects caused by these different Al species. The present study combines the physicochemical processing of three recently studied Al species (metallic Al0, mineral Al2O3, and soluble AlCl3) in artificial digestion fluids with in vitro cell systems for the human intestinal barrier. Inductively coupled plasma mass spectrometry (ICP-MS) and small-angle X-ray scattering (SAXS) methods were used to characterize the Al species in the artificial digestion fluids and in cell culture medium for proliferating and differentiated intestinal Caco-2 cells. Cytotoxicity testing and cellular impedance measurements were applied to address the effects of digested Al species on cell viability and cell proliferation. Microarray-based transcriptome analyses and quantitative real-time PCR were conducted to obtain a deeper insight into cellular mechanisms of action and generated indications for cellular oxidative stress and an influence on xenobiotic metabolism, connected with alterations in associated signaling pathways. These cellular responses, which were predominantly caused by formerly ionic Al species and only at very high concentrations, were not impacted by artificial digestion. A two-directional conversion of Al between ionic species and solid particles occurred throughout all segments of the gastrointestinal tract, as evidenced by the presence of nanoscaled particles. Nevertheless, this presence did not increase the toxicity of the respective Al species.
ZnO nanoparticles are found in different food and consumer products, and their toxicological effects are still under investigation. It is therefore important to understand their behavior in the gastrointestinal tract. Here, we used an in vitro model to assess the physicochemical fate of ZnO nanoparticles during the digestive process in artificial saliva, stomach juice, and intestinal juice. Atomic absorption spectrometry and small-angle X-ray scattering were employed to investigate two ZnO nanomaterials, one intensively characterized reference material and soluble ZnCl2 in a broad range of concentrations between 25 and 1000 μg/mL in the intestinal fluid. Because food components may influence the behavior of nanomaterials in the gastrointestinal tract, starch, milk powder, and olive oil were used to mimic carbohydrates, protein, and fat, respectively. Additionally, ion release of all Zn species was assessed in cell culture media and compared to artificial intestinal juice to investigate relevance of typical cell culture conditions in ZnO nanotoxicology. ZnCl2 as well as the ZnO species were present as particles in artificial saliva but were solubilized completely in the acidic stomach juice. Interestingly, in the intestinal fluid a concentration-independent de novo formation of particles in the nanoscale range was shown. This was the case for all particles as well as for ZnCl2, regardless of the concentration used. Neither of the food components affected the behavior of any Zn species. On the contrary, all Zn species showed a Zn-concentration-dependent ion release in common cell culture medium. This questions the suitability of cell culture studies to investigate the effect of ZnO nanoparticles on intestinal cells. Our results show that Zn-containing nanoparticles reach the intestine. This underlines the importance of determining the influence of the test environment on nanoparticle fate.
Because of the rising application of nanoparticles in food and food-related products, we investigated the influence of the digestion process on the toxicity and cellular uptake of silver nanoparticles for intestinal cells. The main food components – carbohydrates, proteins and fatty acids – were implemented in an in vitro digestion process to simulate realistic conditions. Digested and undigested silver nanoparticle suspensions were used for uptake studies in the well-established Caco-2 model. Small-angle X-ray scattering was used to estimate particle core size, size distribution and stability in cell culture medium. Particles proved to be stable and showed radii from 3.6 to 16.0 nm. Undigested particles and particles digested in the presence of food components were comparably taken up by Caco-2 cells, whereas the uptake of particles digested without food components was decreased by 60%. Overall, these findings suggest that in vivo ingested poly (acrylic acid)-coated silver nanoparticles may reach the intestine in a nanoscaled form even if enclosed in a food matrix. While appropriate for studies on the uptake into intestinal cells, the Caco-2 model might be less suited for translocation studies. Moreover, we show that nanoparticle digestion protocols lacking food components may lead to misinterpretation of uptake studies and inconclusive results.
Size and shape are crucial parameters which have impact on the potential of nanoparticles to penetrate cell membranes and epithelial barriers. Current research in nanotoxicology additionally focuses on particle coating. To distinguish between core- and coating-related effects in nanoparticle uptake and translocation, two nanoparticles equal in size, coating and charge but different in core material were investigated.
Silver and iron oxide nanoparticles coated with poly(acrylic acid) were chosen and extensively characterized by small-angle x-ray scattering, nanoparticle tracing analysis and transmission electron microscopy (TEM). Uptake and transport were studied in the intestinal Caco-2 model in a Transwell System with subsequent elemental analysis. TEM and ion beam microscopy were conducted for particle visualization.
Although equal in size, charge and coating, the behavior of the two particles in Caco-2 cells was different: while the internalized amount was comparable, only iron oxide nanoparticles additionally passed the epithelium. Our findings suggest that the coating material influenced only the uptake of the nanoparticles whereas the translocation was determined by the core material.
Knowledge about the different roles of the particle coating and core materials in crossing biological barriers will facilitate toxicological risk assessment of nanoparticles and contribute to the optimization of pharmacokinetic properties of nano-scaled pharmaceuticals.