Incompatibility of resazurin to detect viable cells in lignin biomaterials: a cautionary study

The nanoparticles prepared from lignin polymer and its methacrylated derivative have gained significant attention in the biomedical field owing to their superior biosafety profile. Here, this study aims towards the synthesis of the methacrylated lignin nanoparticles (MLNPs) using the nanoprecipitation method and understanding their biocompatibility in A549 cells using the resazurin assay. In our study, the nanoprecipitation method yielded a stable suspension of MLNPs having sub-200 nm size. When incubated with A549 cells, initially, these nanoparticles showed cytotoxicity and reduced biocompatibility from the well-known resazurin assay. However, careful microscopic examination of the cells showed healthy and well-adhered A549 cells with normal cobblestone-like epithelial morphology. Hence, we performed a detailed analysis of MLNPs to understand their interaction with resazurin with respect to concentration and time while keeping pristine lignin nanoparticles (LNPs) as a control. We found that LNPs and MLNPs interfered with resazurin assay, leading to false-positive results. Our study highlights a significant drawback in the use of resazurin assay for these nanoparticles and emphasizes the critical need for alternative approaches to evaluate their biocompatibility. In an attempt to find alternative, conventional MTT assay offered highly reproducible biocompatibility results without any interference during evaluating the cytotoxicity of these nanoparticles.