Proteomic profiling reveals engineered chitosan nanoparticles mediated cellular crosstalk and immunomodulation for therapeutic application in apical periodontitis

Macrophages (MQ) are major constituents of chronically inflamed periapical tissues in apical periodontitis. This study aimed to investigate the immunomodulatory effect of engineered bioactive chitosan-based nanoparticles (CSnp) antibiofilm medication on MQ cocultured with periodontal ligament fibroblasts (PdLF). Cells viability, spreading, PdLF migration, and intracellular CSnp uptake were characterized. Tandem Mass Tag-based proteomics was applied to analyze MQ global protein expression profiles after interaction with Enterococcus faecalis biofilm, CSnp-treated biofilm, and CSnp. Secreted inflammatory mediators were analyzed. Following bioinformatics analyses, candidate proteins were validated via targeted proteomics. CSnp maintained cells viability, increased MQ spreading, and PdLF migration (p < 0.05). Transmission electron micrographs demonstrated CSnp internalization via macropinocytosis, clathrin-mediated endocytosis, and phagocytosis. Proteomic analysis revealed that CSnp-treated biofilm upregulated proteins (>1.5-folds, p < 0.05) showed functional enrichment in the pathway of metal sequestration by antimicrobial proteins, while downregulated proteins showed enrichment in ferroptosis. CSnp upregulated proteins exhibiting antioxidant and immunoregulatory properties. Upregulation of SERPINB1 by CSnp (>1.5-folds, p < 0.05) was validated. CSnp-treated biofilm reduced pro-inflammatory IL-1β and nitric oxide but enhanced anti-inflammatory IL-10 and TGF-β1 (p < 0.05). Internalized engineered bioactive CSnp reprogrammed MQ proteomic and cytokine profiles to modulate biofilm-mediated inflammation, and prompted PdLF migration, emphasizing its potential to regulate healing process in the treatment of apical periodontitis.