Christoph Bantz

Inflammatory and cytotoxic responses of an alveolar-capillary coculture model to silica nanoparticles: Comparison with conventional monocultures

BackgroundTo date silica nanoparticles (SNPs) play an important role in modern technology and nanomedicine. SNPs are present in various materials (tyres, electrical and thermal insulation material, photovoltaic facilities). They are also used in products that are directly exposed to humans such as cosmetics or toothpaste. For that reason it is of great concern to evaluate the possible hazards of these engineered particles for human health. Attention should primarily be focussed on SNP effects on biological barriers. Accidentally released SNP could, for example, encounter the alveolar-capillary barrier by inhalation. In this study we examined the inflammatory and cytotoxic responses of monodisperse amorphous silica nanoparticles (aSNPs) of 30 nm in size on an in vitro coculture model mimicking the alveolar-capillary barrier and compared these to conventional monocultures.MethodsThus, the epithelial cell line, H441, and the endothelial cell line, ISO-HAS-1, were used in monoculture and in coculture on opposite sides of a filter membrane. Cytotoxicity was evaluated by the MTS assay, detection of membrane integrity (LDH release), and TER (Transepithelial Electrical Resistance) measurement. Additionally, parameters of inflammation (sICAM-1, IL-6 and IL-8 release) and apoptosis markers were investigated.ResultsRegarding toxic effects (viability, membrane integrity, TER) the coculture model was less sensitive to apical aSNP exposure than the conventional monocultures of the appropriate cells. On the other hand, the in vitro coculture model responded with the release of inflammatory markers in a much more sensitive fashion than the conventional monoculture. At concentrations that were 10-100fold less than the toxic concentrations the apically exposed coculture showed a release of IL-6 and IL-8 to the basolateral side. This may mimic the early inflammatory events that take place in the pulmonary alveoli after aSNP inhalation. Furthermore, a number of apoptosis markers belonging to the intrinsic pathway were upregulated in the coculture following aSNP treatment. Analysis of the individual markers indicated that the cells suffered from DNA damage, hypoxia and ER-stress.ConclusionWe present evidence that our in vitro coculture model of the alveolar-capillary barrier is clearly advantageous compared to conventional monocultures in evaluating the extent of damage caused by hazardous material encountering the principle biological barrier in the lower respiratory tract.

High-resolution investigation of nanoparticle interaction with a model pulmonary surfactant monolayer.

The pulmonary surfactant film spanning the inner alveolar surface prevents alveolar collapse during the end-exhalation and reduces the work of breathing. Nanoparticles (NPs) present in the atmosphere or nanocarriers targeted through the pulmonary route for medical purposes challenge this biological barrier. During interaction with or passage of NPs through the alveolar surfactant, the biophysical functioning of the film may be altered. However, experimental evidence showing detailed biophysical interaction of NPs with the pulmonary surfactant film are scant. In this study, we have investigated the impact of a hydrophobic polyorganosiloxane (AmOrSil20) NPs on the integrity as well as on the structural organization of the model pulmonary surfactant film. Primarily, scanning force microscopic techniques and electron microscopy have been used to visualize the topology as well as to characterize the localization of nanoparticles within the compressed pulmonary surfactant film. We could show that the NPs partition in the fluid phase of the compressed film at lower surface pressure, and at higher surface pressure, such NPs interact extensively with the surface-associated structures. Major amounts of NPs are retained at the interface and are released slowly into the aqueous subphase during repeated compression/expansion cycles. Further, the process of vesicle insertion into the interfacial film was observed to slow down with increasing NP concentrations. The hydrophobic AmOrSil20 NPs up to a given concentration do not substantially affect the structural organization and functioning of pulmonary surfactant film; however, such NPs do show drastic impacts at higher concentrations.

The protein corona protects against size- and dose-dependent toxicity of amorphous silica nanoparticles.

Summary Besides the lung and skin, the gastrointestinal (GI) tract is one of the main targets for accidental exposure or biomedical applications of nanoparticles (NP). Biological responses to NP, including nanotoxicology, are caused by the interaction of the NP with cellular membranes and/or cellular entry. Here, the physico-chemical characteristics of NP are widely discussed as critical determinants, albeit the exact mechanisms remain to be resolved. Moreover, proteins associate with NP in physiological fluids, forming the protein corona potentially transforming the biological identity of the particle and thus, adding an additional level of complexity for the bio–nano responses. Here, we employed amorphous silica nanoparticles (ASP) and epithelial GI tract Caco-2 cells as a model to study the biological impact of particle size as well as of the protein corona. Caco-2 or mucus-producing HT-29 cells were exposed to thoroughly characterized, negatively charged ASP of different size in the absence or presence of proteins. Comprehensive experimental approaches, such as quantifying cellular metabolic activity, microscopic observation of cell morphology, and high-throughput cell analysis revealed a dose- and time-dependent toxicity primarily upon exposure with ASP30 (Ø = 30 nm). Albeit smaller (ASP20, Ø = 20 nm) or larger particles (ASP100; Ø = 100 nm) showed a similar zeta potential, they both displayed only low toxicity. Importantly, the adverse effects triggered by ASP30/ASP30L were significantly ameliorated upon formation of the protein corona, which we found was efficiently established on all ASP studied. As a potential explanation, corona formation reduced ASP30 cellular uptake, which was however not significantly affected by ASP surface charge in our model. Collectively, our study uncovers an impact of ASP size as well as of the protein corona on cellular toxicity, which might be relevant for processes at the nano–bio interface in general.

In vitro investigation of silica nanoparticle uptake into human endothelial cells under physiological cyclic stretch

BackgroundIn general the prediction of the toxicity and therapeutic efficacy of engineered nanoparticles in humans is initially determined using in vitro static cell culture assays. However, such test systems may not be sufficient for testing nanoparticles intended for intravenous application. Once injected, these nanoparticles are caught up in the blood stream in vivo and are therefore in continuous movement. Physical forces such as shear stress and cyclic stretch caused by the pulsatile blood flow are known to change the phenotype of endothelial cells which line the luminal side of the vasculature and thus may be able to affect cell-nanoparticle interactions.MethodsIn this study we investigated the uptake of amorphous silica nanoparticles in primary endothelial cells (HUVEC) cultured under physiological cyclic stretch conditions (1 Hz, 5% stretch) and compared this to cells in a standard static cell culture system. The toxicity of varying concentrations was assessed using cell viability and cytotoxicity studies. Nanoparticles were also characterized for the induction of an inflammatory response. Changes to cell morphology was evaluated in cells by examining actin and PECAM staining patterns and the amounts of nanoparticles taken up under the different culture conditions by evaluation of intracellular fluorescence. The expression profile of 26 stress-related was determined by microarray analysis.ResultsThe results show that cytotoxicity to endothelial cells caused by silica nanoparticles is not significantly altered under stretch compared to static culture conditions. Nevertheless, cells cultured under stretch internalize fewer nanoparticles. The data indicate that the decrease of nanoparticle content in stretched cells was not due to the induction of cell stress, inflammation processes or an enhanced exocytosis but rather a result of decreased endocytosis.ConclusionsIn conclusion, this study shows that while the toxic impact of silica nanoparticles is not altered by stretch this dynamic model demonstrates altered cellular uptake of nanoparticles under physiologically relevant in vitro cell culture models. In particular for the development of nanoparticles for biomedical applications such improved in vitro cell culture models may play a pivotal role in the reduction of animal experiments and development costs.

Flotillin-involved uptake of silica nanoparticles and responses of an alveolar-capillary barrier in vitro

Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60 μg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560±8 Ω cm(2). H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake.

Pulmonary surfactant augments cytotoxicity of silica nanoparticles: Studies on an in vitro air-blood barrier model.

Summary The air–blood barrier is a very thin membrane of about 2.2 µm thickness and therefore represents an ideal portal of entry for nanoparticles to be used therapeutically in a regenerative medicine strategy. Until now, numerous studies using cellular airway models have been conducted in vitro in order to investigate the potential hazard of NPs. However, in most in vitro studies a crucial alveolar component has been neglected. Before aspirated NPs encounter the cellular air–blood barrier, they impinge on the alveolar surfactant layer (10–20 nm in thickness) that lines the entire alveolar surface. Thus, a prior interaction of NPs with pulmonary surfactant components will occur. In the present study we explored the impact of pulmonary surfactant on the cytotoxic potential of amorphous silica nanoparticles (aSNPs) using in vitro mono- and complex coculture models of the air–blood barrier. Furthermore, different surface functionalisations (plain-unmodified, amino, carboxylate) of the aSNPs were compared in order to study the impact of chemical surface properties on aSNP cytotoxicity in combination with lung surfactant. The alveolar epithelial cell line A549 was used in mono- and in coculture with the microvascular cell line ISO-HAS-1 in the form of different cytotoxicity assays (viability, membrane integrity, inflammatory responses such as IL-8 release). At a distinct concentration (100 µg/mL) aSNP–plain displayed the highest cytotoxicity and IL-8 release in monocultures of A549. aSNP–NH2 caused a slight toxic effect, whereas aSNP–COOH did not exhibit any cytotoxicity. In combination with lung surfactant, aSNP–plain revealed an increased cytotoxicity in monocultures of A549, aSNP–NH2 caused a slightly augmented toxic effect, whereas aSNP–COOH did not show any toxic alterations. A549 in coculture did not show any decreased toxicity (membrane integrity) for aSNP–plain in combination with lung surfactant. However, a significant augmented IL-8 release was observed, but no alterations in combination with lung surfactant. The augmented aSNP toxicity with surfactant in monocultures appears to depend on the chemical surface properties of the aSNPs. Reactive silanol groups seem to play a crucial role for an augmented toxicity of aSNPs. The A549 cells in the coculture seem to be more robust towards aSNPs, which might be a result of a higher differentiation and polarization state due the longer culture period.

Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on Protein Adsorption in Serum and Cellular Uptake.

Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.

Characterization of Nanoparticles Under Physiological Conditions

In this article, well-established characterization methods for nanoparticles (NPs) are discussed, in particular their application under physiological conditions. The impact of different media, mimicking physiological conditions, on NP stability in terms of physiological ionic strength and formation of the NP–protein corona is described. In order to characterize NPs under physiological conditions, we distinguish between scattering and correlation methods, microscopy-based methods, and methods based on hydrodynamic separation. Features and limitations of relevant characterization methods are reviewed, as well as challenges arising in physiological media from enhanced aggregation tendency and the presence of proteins. We conclude that no available method for NP characterization in physiological media is able to describe the colloidal system completely and satisfactory. On the contrary, combining well-chosen analytical methods by taking benefits and disadvantages into account may provide detailed characterization results.