Proinflammatory and cytotoxic response to nanoparticles in precision-cut lung slices

Abstract

SummaryPrecision-cut lung slices (PCLS) are an established ex vivo alternative to in vivo experiments in pharmacotoxicology. The aim of this study was to evaluate the potential of PCLS as a tool in nanotoxicology studies. Silver (Ag-NPs) and zinc oxide (ZnO-NPs) nanoparticles as well as quartz particles were used because these materials have been previously shown in several in vitro and in vivo studies to induce a dose-dependent cytotoxic and inflammatory response. PCLS were exposed to three concentrations of 70 nm monodisperse polyvinylpyrrolidone (PVP)-coated Ag-NPs under submerged culture conditions in vitro. ZnO-NPs (NM110) served as ‘soluble’ and quartz particles (Min-U-Sil) as ‘non-soluble’ control particles. After 4 and 24 h, the cell viability and the release of proinflammatory cytokines was measured. In addition, multiphoton microscopy was employed to assess the localization of Ag-NPs in PCLS after 24 h of incubation. Exposure of PCLS to ZnO-NPs for 4 and 24 h resulted in a strong decrease in cell viability, while quartz particles had no cytotoxic effect. Moreover, only a slight cytotoxic response was detected by LDH release after incubation of PCLS with 20 or 30 µg/mL of Ag-NPs. Interestingly, none of the particles tested induced a proinflammatory response in PCLS. Finally, multiphoton microscopy revealed that the Ag-NP were predominantly localized at the cut surface and only to a much lower extent in the deeper layers of the PCLS. In summary, only ‘soluble’ ZnO-NPs elicited a strong cytotoxic response. Therefore, we suggest that the cytotoxic response in PCLS was caused by released Zn2+ ions rather than by the ZnO-NPs themselves. Moreover, Ag-NPs were predominantly localized at the cut surface of PCLS but not in deeper regions, indicating that the majority of the particles did not have the chance to interact with all cells present in the tissue slice. In conclusion, our findings suggest that PCLS may have some limitations when used for nanotoxicology studies. To strengthen this conclusion, however, other NP types and concentrations need to be tested in further studies.

PVP-coated, negatively charged silver nanoparticles: A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments

“…Details on the experiments with precision-cut lung slices (PCLS) are given in [126]. Details on the triple-cell 3D culture are described in [125,127–129].…”

“…b Significant, but weak increase in LDH release after incubation for 4 h and 24 h. No increase in CXCL-1 or TNF-α protein as determined by ELISA [126]. c Significant increases in BALF LDH, total protein, and cytokine levels as well as in BALF neutrophil numbers 24 h after intratracheal instillation of 250 µg silver nanoparticles [124].…”

Alternative Testing Strategies for Nanomaterials: State of the Science and Considerations for Risk Analysis

“…Comparison of ex vivo and in vivo gut exposure to TiO 2 NM show similar modes of translocation and localization in the epithelium of both models . Rat, mouse, or human precision‐cut lung slices are used to assess toxicant effects on the respiratory tract, such as cytotoxicity, genotoxicity, altered protein content, oxidative stress, apoptosis, cytokine release, and histological changes . The use of ex vivo methods for NM is still uncertain due to susceptibility to interferences, highly variable results, and an inability to determine appropriate dose metrics that relate to in vivo exposure .…”

“…The use of ex vivo methods for NM is still uncertain due to susceptibility to interferences, highly variable results, and an inability to determine appropriate dose metrics that relate to in vivo exposure . Ex vivo methods may be useful for prioritization and ranking of NM toxicity but further method optimization is likely needed to address reproducibility, false positives, false negatives, and to better understand the limitations of the system . Despite these limitations, the use of other ex vivo methods for NM still provides critical information on end points such as distribution, penetration, and uptake, and remains a promising method for testing broader types of NM.…”

In vitro-ex vivo model systems for nanosafety assessment

“…Although most studies have focused on aspects of pharmatoxicology (44,45), some recent publications have proven that PCS-based approaches are highly relevant for the risk assessment of nanomaterials and xenobiotics in general, in terms of inflammation, organ injury and sensitization (46)(47)(48). A recently published study showed, however, that the system might only be useful for ions released from nanoparticles or soluble substances since it was observed that the silver nanoparticles did predominantly attach at the cut surfaces of the PCS from lung tissues ( Figure 1C) but could hardly penetrate into deeper regions (49).…”