Abstract
<title>Abstract</title> <p>While protein fibrillation represents a critical challenge in therapeutic insulin delivery, the structural determinants and immunological consequences of insulin-derived fibrils (IDFs) formed in the presence of phenolic preservatives remain poorly characterized. This study investigated the structural characteristics of IDFs formed with (IDF (+)) and without (IDF (-)) phenolic preservatives and elucidated their differential immunomodulatory mechanisms in bone marrow-derived macrophages (BMDMs). IDF structural properties were characterized using Thioflavin T fluorescence and nanoparticle tracking analysis (Spectradyne nCS1™). BMDMs were treated with IDF (+), IDF (-), or m-cresol. Cytotoxicity, reactive oxygen species (ROS) production, MIP-1α levels, and expression of signaling pathways were quantified. Structural analysis revealed similar aggregation states between IDF (+) and IDF (-). However, IDF (+) induced greater cytotoxicity and ROS production than IDF (-), which produced minimal ROS. Both fibrils increased MIP-1α levels. Additionally, IDF (-) upregulated NRF2 whereas m-cresol downregulated STAT6. Together, these results suggest distinct mechanisms of macrophage activation and suggest that protein aggregates can induce macrophage responses directly. Insulin fibrils activate macrophage inflammatory pathways through ROS-independent mechanisms. Phenolic preservatives enhance fibril cytotoxicity and likely ROS production while differentially modulating inflammatory signaling. These findings suggest that strategies to attenuate the effects of IDFs in insulin infusion therapy may increase longevity and biocompatibility of these devices.</p>