Tracing micro and nanoplastics toxicity in human pulmonary fibroblasts through integrated Raman and transcriptomic analyses.

Inhaled micro- and nanoplastics can reach the distal regions of the lungs, where their elimination is limited due to the lack of efficient clearance mechanisms. Although polystyrene particles have been detected in human lung tissue, the molecular effects of such exposures remain poorly characterized. Understanding the cellular response to microplastics exposure, particularly at the transcriptional and structural levels, is essential for assessing potential health risks. The purpose of this study was to evaluate the impact of primary polystyrene micro- and nanoparticles on human pulmonary fibroblasts, a relevant in vitro model for investigating the molecular mechanisms underlying microplastics-induced pulmonary toxicity. Monodisperse polystyrene particles with diameters of 0.1, 1 and 5 μm were used to evaluate size-dependent internalization and cellular response in human pulmonary fibroblasts. Cells were cultured under standard conditions and exposed to particles in vitro. Internalization and fate of microplastics were tested using Raman microscopy, while transcriptomic alterations were assessed by RNA sequencing to identify early molecular responses associated with particle size. Raman microscopy confirmed the internalization of 0.1 μm polystyrene particles by human pulmonary fibroblasts. Particles sized 1 μm showed a high affinity for the fibroblast cell membrane, however, definitive confirmation or exclusion of their internalization into the cells was not possible due to the sample preparation protocol and measurement conditions used. In contrast, exposure to 5 μm particles resulted in pronounced cytotoxicity across tested concentrations, precluding RNA-seq analysis. Transcriptomic profiling assessed by principal component analysis revealed distinct gene expression patterns in cells following exposure to 0.1 and 1 μm particles. Exposure to 0.1 μm particles led to upregulation of genes involved in mitochondrial function and protein synthesis. In contrast, 1 μm particles caused downregulation of genes associated with oxidative phosphorylation and proteostasis. This study shows that particle size and concentration critically influence the molecular response of human pulmonary fibroblasts to polystyrene micro- and nanoparticles. Raman microscopy proved a valuable tool for detecting particle internalization and assessing size-related biochemical changes, including in the nanoscale range.