Dataset Information

Determination of Silver Nanoparticle Dose in vitro.

ABSTRACT: An important issue for interpreting in vitro nanomaterial testing is quantifying the dose delivered to target cells. Considerations include the concentration added to the culture, the proportion of the applied dose that interacts with the target cells, and the amount that is eventually absorbed by the target cells. Rapid and efficient techniques are needed to determine delivered doses. Previously, we demonstrated that TiO₂ and silver nanoparticles (AgNP) were absorbed by cells in a dose dependent manner between 1 μg/ml and 30 μg/ml and were detected in cells by light scatter using a flow cytometer. Here, we compare four potential indices of the dose of AgNP to cells, including: inductively coupled plasma - mass spectrometry (ICP-MS); flow cytometry side scatter (SSC); and amount of silver deposited to the cell layer as estimated with both an integrated Volumetric Centrifugation Method - In Vitro Sedimentation, Diffusion and Dosimetry Model (VCM-ISDD) and a Distorted Grid (DG) model. A retinal pigment epithelial cell line was exposed to 20 nm or 75 nm citrate-coated AgNP for 24 hr. The relationships between particle sizes and internalized doses varied according to the dose metric. Twenty-four hours after exposure, the cell layer contained a greater mass of silver when treated with 75 nm AgNP than with 20 nm AgNP. When the dose was expressed as the number of particles or as the total surface area of absorbed particles, however, the reverse was true; the dose to the cells was higher after exposure to 20 than 75 nm AgNP. Flow cytometry SSC increased with dose for both sizes of AgNP, and was correlated with Ag in cells measured by ICP-MS. The rate of SSC increase was greater for 75 than for 20 nm AgNP, suggesting it could be used as an indicator of cellular dose after accounting for particle size and composition. Silver was detected by ICP-MS in re-suspended supernates of the isolated cell layer suggested that not all the silver deposited to the cell layer was absorbed by the cells. Both the VCM-ISDD and DG models estimated the proportion of Ag deposited to the cellular layer, which in both cases was greater than the amount of silver in the cells measured by ICP-MS. Modeled deposition more closely compared to the total Ag deposition by ICP-MS, i.e. mass of silver in the cells plus the resuspended, unabsorbed Ag from the cell layer. ICP-MS indicated the mass of silver in cells from AgNP treatment, but not whether the Ag was in the form of particles or dissolved ions. Deposition models predicted the amount of AgNP deposited to the cell layer, but not cellular uptake. Flow cytometry SSC was correlated to cellular uptake of particle-form AgNP and could be calibrated against ICP-MS to indicate mass of cellular uptake. Therefore, a combination of approaches may be required to accurately understand cellular dosimetry of in vitro nanotoxicology experiments. In summary, cellular dosimetry is an important consideration for nanotoxicology experiments, and not necessarily related to the applied dose.

SUBMITTER: Ortenzio J

PROVIDER: S-EPMC8312577 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Spontaneous agglomeration of engineered nanoparticles (ENPs) is a common problem in cell culture media which can confound interpretation of in vitro nanotoxicity studies. The authors created stable agglomerates of iron oxide nanoparticles (IONPs) in conventional culture medium, which varied in hydrodynamic size (276 nm-1.5 ?m) but were composed of identical primary particles with similar surface potentials and protein coatings. Studies using C10 lung epithelial cells show that the dose rate effects of agglomeration can be substantial, varying by over an order of magnitude difference in cellular dose in some cases. Quantification by magnetic particle detection showed that small agglomerates of carboxylated IONPs induced greater cytotoxicity and redox-regulated gene expression when compared with large agglomerates on an equivalent total cellular IONP mass dose basis, whereas agglomerates of amine-modified IONPs failed to induce cytotoxicity or redox-regulated gene expression despite delivery of similar cellular doses. Dosimetry modelling and experimental measurements reveal that on a delivered surface area basis, large and small agglomerates of carboxylated IONPs have similar inherent potency for the generation of ROS, induction of stress-related genes and eventual cytotoxicity. The results suggest that reactive moieties on the agglomerate surface are more efficient in catalysing cellular ROS production than molecules buried within the agglomerate core. Because of the dynamic, size and density-dependent nature of ENP delivery to cells in vitro, the biological consequences of agglomeration are not discernible from static measures of exposure concentration (?g/ml) alone, highlighting the central importance of integrated physical characterisation and quantitative dosimetry for in vitro studies. The combined experimental and computational approach provides a quantitative framework for evaluating relationships between the biocompatibility of nanoparticles and their physical and chemical characteristics.

Project description:The application of engineered silver nanoparticles (AgNPs) in a considerable amount of registered commercial products inevitably will result in the continuous release of AgNPs into the natural aquatic environment. Therefore, native biofilms, as the prominent life form of microorganisms in almost all known ecosystems, will be subjected to AgNP exposure. Despite the exponentially growing research activities worldwide, it is still difficult to assess nanoparticle-mediated toxicity in natural environments. In order to obtain an ecotoxicologically relevant exposure scenario, we performed experiments with artificial stream mesocosm systems approaching low dose AgNP concentrations close to predicted environmental concentrations. Pregrown freshwater biofilms were exposed for 14 days to citrate-stabilized AgNPs at a concentration of 600 ?g l-1 in two commonly used sizes (30 and 70 nm). Sublethal effects of AgNP treatment were assessed with regard to biofilm structure by gravimetric measurements (biofilm thickness and density) and by two biomass parameters, chlorophyll a and protein content. The composition of bacterial biofilm communities was characterized by t-RFLP fingerprinting combined with phylogenetic studies based on the 16S gene. After 14 days of treatment, the structural parameters of the biofilm such as thickness, density, and chlorophyll a and protein content were not statistically significantly changed by AgNP exposure. Furthermore, t-RFLP fingerprint analysis showed that the bacterial diversity was not diminished by AgNPs, as calculated by Shannon Wiener and evenness indices. Nevertheless, t-RFLP analysis also indicated that AgNPs led to an altered biofilm community composition as was shown by cluster analysis and multidimensional scaling (MDS) based on the Bray Curtis index. Sequence analysis of cloned 16S rRNA genes further revealed that changes in community composition were related with the displacement of putatively AgNP-sensitive bacterial taxa Actinobacteria, Chloroflexi, and Cyanobacteria by taxa known for their enhanced adaptability towards metal stress, such as Acidobacteria, Sphingomonadales, and Comamonadaceae. This measurable community shift, even after low dose AgNP treatment, causes serious concerns with respect to the broad application of AgNPs and their potentially adverse impact on the ecological function of lotic biofilms, such as biodegradation or biostabilization.

Dataset Information

Determination of Silver Nanoparticle Dose in vitro.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets