Pulmonary inflammation and immune responses induced by nanocellulose: Insights from in vivo and in vitro models

The increasing use of nanocellulose (NC), including cellulose nanofibrils (CNFs) and cellulose nanocrystals (CNCs), in industrial, biomedical, and consumer products has raised concerns regarding potential inhalation exposure, as these materials contain components within the respirable particle size range (<10 µm). Despite expanding applications, data on NC-induced pulmonary and systemic immune effects remain limited. This study investigated the pulmonary and immunotoxic effects of CNF1 (TEMPO-oxidized), CNF2 (mechanically fibrillated), and CNC1 in rats following intratracheal instillation, using multi-walled carbon nanotubes (MWCNTs) as a benchmark. All materials were administered at 2.0 mg/kg body weight, with fiber diameters of 14.1-28.2 nm and lengths of 0.7-2.2 µm. At 28 d post-instillation, all NC were phagocytosed by alveolar macrophages. Bronchoalveolar lavage fluid (BALF) and histopathological analyses revealed that CNF2 induced limited inflammation with granuloma formation and minimal BALF changes, whereas CNF1 and CNC1 triggered similar changes in BALF inflammatory markers. Although CNC1 elicited the most notable histopathological changes among NCs, all NC-induced responses were less severe than those caused by MWCNTs. No significant alterations were observed in lymphocyte subsets in the spleen or thymus, indicating minimal systemic immunotoxicity. <i>In vitro</i> assays using NR8383 alveolar macrophages were performed to compare CNC1, sulfuric acid-hydrolyzed CNC2, and desulfurized CNC3. All CNCs were internalized and stimulated pro-inflammatory cytokine production, with responses influenced by surface chemistry despite similar size and morphology. CNC1 and CNC2 exhibited low cytotoxicity following 48h exposure at concentrations up to 100 μg/mL. In contrast, CNC3 induced mild to moderate cytotoxicity under the same conditions, upregulated genes linked to inflammatory responses, oxidative stress, apoptosis, and extracellular matrix degradation. These findings reveal that NCs generally exhibit lower pulmonary toxicity than MWCNTs; however, their biological effects are strongly modulated by fiber morphology, surface characteristics, and deposition behavior. To ensure safe use of NCs, comprehensive, material-specific toxicity assessments and standardized evaluation frameworks are essential. In particular, rigorous endotoxin testing and control should be incorporated into future studies to maintain validity and reproducibility of hazard evaluations. Additionally, long-term and repeated exposure models, mechanistic investigations, and case-by-case safety assessments are required, especially for NC variants with limited biodegradability and prolonged pulmonary retention.