Protocol for determination of IC50 of Advanced Nanomaterials and nano-microplastics in vitro using monocultures of cells v1

Monoculture of cells under submerged conditions allows high-throughput screening due to its cost-effectiveness and ease of use. Since the lung and the gastrointestinal tract have been identified as high concerns for advanced nanomaterials (Ad-NMs), two representative cell lines were selected to assess toxicity in this protocol. The A549 cell line, a human epithelial cell line derived from lung carcinoma and commonly used as a model of the alveolar type II epithelial cell, was used for the lung. The Caco-2 cell line, a human epithelial colorectal adenocarcinoma cell line commonly used as a model of the intestinal barrier for drug absorption and transport studies, was used for the intestinal counterpart. The same time points and assays for viability assessment were used for both cell lines to develop a generic protocol for in vitro toxicity testing. Ad-NMs are known to interfere with viability assays due to their large surface areas and chemically active surfaces, potentially leading to false toxicity assessments. This protocol uses graphene oxide nanoparticles with different oxidation states and flake sizes (GO1-GO4) as case material. Several assays based on different principles were selected, including MTS metabolic conversion, LDH release, and ATP release, to determine the most suitable assays for assessing the toxicity of Ad-NMs. In addition, a positive control for cytotoxicity (Triton X-100) with particles at all tested concentrations was added to determine if the particles mask any cellular effects. A blank (medium only, without cells) was also added to see whether the particles had any fluorescent or luminescent background. Results: No dose-response curves were obtained for A549 and Caco2 cells exposed to the particles GO1, GO2, GO3, and GO4 using the MTS and LDH release assay (Figures 3 and 5) while dose-response curves were observed in A549 cells using the ATP release assay (Figure 4). Controls exposed to both the particles and Triton X-100 did not show any interference, which indicates that the particles do not mask the assays themselves, confirming the validity of the assay in detecting changes.