The shape and durability of inhalable fibers is connected with their potential to cause lung cancer. Especially toxicological effects of nanofibers and their derivatives are still incompletely understood. Currently, their safety evaluation is performed under in vivo settings. However, as nanofiber applications continue to expand, alternative approaches are urgently needed that align with the 3 R principles (Replacement, Reduction, Refinement). To this end, silicon carbide (SiC) and titanium dioxide (TiO₂) nanofibers as well as their ground fragments were tested in the NR8383 alveolar macrophage assay. Intact nanofibers induced dose-dependent cytotoxicity, oxidative stress, and the release of pro-inflammatory cytokines, while their ground counterparts elicited minimal effects. The subsequent proteomic profiling of cells exposed to a sub-cytotoxic nanofiber concentration revealed significant alterations in the levels of 32 % (SiC) and 8 % (TiO₂) of all detected proteins compared to untreated cells. Besides protein modifications induced by oxidative stress, key alterations comprised protein clusters attributed to inflammation (n_proteins=9), vesicular trafficking (n = 22), metabolic changes (n = 32) and apoptosis (n = 5). Cells treated with equal amounts of ground nanofibers exhibited only negligible changes, highlighting the morphology-driven nature of the effects. Finally, a set of 58 proteins are proposed as a proteomic fingerprint of nanofiber-related toxicity at the cellular level. Overall, the study substantiates fiber morphology-driven effects of nanofibers in alveolar macrophages and outlines concrete protein biomarkers to describe nanofiber pathogenicity along with underlying mechanisms. This work contributes to the development of a robust in vitro testing strategy required for the Safe-and-Sustainable-by-Design demand of the European Commission.

This article is published in the Journal "Nano Today" (2025).