Comparative proteomic analysis of hepatic effects induced by nanosilver, silver ions and nanoparticle coating in rats.

The presence of nano-scaled particles in food and food-related products has drawn attention to the oral uptake of nanoparticles and their interactions with biological systems. In the present study, we used a toxicoproteomics approach to allow for the untargeted experimental identification and comparative analysis of cellular responses in rat liver after repeated-dose treatment with silver nanoparticles, ions, and the coating matrix used for particle stabilization. The proteomic analysis revealed treatment-related effects caused by exposure to silver in particulate and ionic form. Both silver species induced similar patterns of signaling and metabolic alterations. Silver-induced cellular alterations comprised, amongst others, proteins involved in metal homeostasis, oxidative stress response, and energy metabolism. However, we discovered that secondary nano-scaled structures were formed from ionic silver. Furthermore, also the coating matrix alone gave rise to the formation of nano-scaled particles. The present data confirm, complement, and extend previous knowledge on silver toxicity in rodent liver by providing a comprehensive proteomic data set. The observation of secondary particle formation from non-particle controls underlines the difficulties in separating particle-, ion-, and matrix coating-related effects in biological systems. Awareness of this issue will support proper evaluation of nanotoxicology-related data in the future.

Comparative proteomic analysis of silver nanoparticle effects in human liver and intestinal cells.

Consumers are orally exposed to nanoparticulate or soluble species of the non-essential element silver due to its use in food contact materials or as a food additive. Potential toxicity of silver nanoparticles has gained special scientific attention. A fraction of ingested ionic or particulate silver is taken up in the intestine and transported to the liver, where it may induce oxidative stress and elicit subsequent adverse responses. Here, we present a comprehensive analysis of global proteomic changes induced in human Hep G2 hepatocarcinoma cells by different concentrations of AgPURE silver nanoparticles or by corresponding concentrations of ionic silver. Bioinformatic analysis of proteomic data confirms and substantiates previous findings on silver-induced alterations related to redox stress, mitochondrial dysfunction, intermediary metabolism, inflammatory responses, posttranslational protein modification and other cellular parameters. Similarities between the effects exerted by the two silver species are in line with the assumption that silver ions released from nanoparticles substantially contribute to their toxicity. Moreover, a comparative bioinformatic evaluation of proteomic effects in hepatic and intestinal cells exerted either by silver nanoparticles or bionic silver is presented. Our results show that, despite remarkable differences at the level of affected proteins in the different cell lines, highly similar biological consequences, corresponding to previous in vivo findings, can be deduced by applying appropriate bioinformatic data mining.

Protein Corona Analysis of Silver Nanoparticles Links to Their Cellular Effects.

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.

Dosimetric Quantification of Coating-Related Uptake of Silver Nanoparticles.

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.

It takes more than a coating to get nanoparticles through the intestinal barrier in vitro.

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.

In vivo distribution of nanosilver in the rat: The role of ions and de novo-formed secondary particles.

Silver nanoparticles are advertised as antimicrobial agents in a wide range of products. The majority of available studies suggest that silver nanoparticle toxicity is mainly caused by silver ions released from the particles. However, it remains challenging to distinguish between the effect of silver nanoparticles and silver ions. Here we used a combination of a short-term in vivo study in rats and an in silico-based toxicokinetic model to determine tissue distribution of administered ionic and nanoparticulate silver, and to estimate mixture ratios of the different silver species, namely primary nanoparticles, ions and secondary particles. Our data indicate that silver nanoparticles and silver ions are not or only marginally bioavailable after oral ingestion of a single, non-toxic dose. Experimental data on organ distribution after intravenous injection were accurately reflected by the predictions of the in silico model. Toxicokinetic modeling suggests systemic distribution of a major proportion of the injected ionic silver as de novo formed secondary nanoparticles, and the presence of such particles was proven by electron microscopy. The observation that silver ions form secondary particles, underlines the difficulties in distinguishing between particle- and ion-dependent effects of silver nanoparticles.

Impact of food components during in vitro digestion of silver nanoparticles on cellular uptake and cytotoxicity in intestinal cells.

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.

Molecular mechanism of silver nanoparticles in human intestinal cells.

Silver nanoparticles are used in consumer products like food contact materials, drinking water technologies and supplements, due to their antimicrobial properties. This leads to an oral uptake and exposure of intestinal cells. In contrast to other studies we found no apoptosis induction by surfactant-coated silver nanoparticles in the intestinal cell model Caco-2 in a previous study, although the particles induced oxidative stress, morphological changes and cell death. Therefore, this study aimed to analyze the molecular mechanism of silver nanoparticles in Caco-2 cells. We used global gene expression profiling in differentiated Caco-2 cells, supported by verification of the microarray data by quantitative real-time RT-PCR and microscopic analysis, impedance measurements and assays for apoptosis and oxidative stress. Our results revealed that surfactant-coated silver nanoparticles probably affect the cells by outside-in signaling. They induce oxidative stress and have an influence on canonical pathways related to FAK, ILK, ERK, MAPK, integrins and adherence and tight junctions, thereby inducing transcription factors like AP1, NFkB and NRF2, which mediate cellular reactions in response to oxidative stress and metal ions and induce changes in the cytoskeleton and cell-cell and cell-matrix contacts. The present data confirm the absence of apoptotic cell death. Non-apoptotic, necrotic cell death, especially in the intestine, can cause inflammation and influence the mucosal immune response.