Pulmonary inflammatory responses and retention dynamics of cellulose nanofibrils
Authors: Fujita Katsuhide , Obara Sawae , Maru Junko , Kawai Yuka , Endoh Shigehisa 
Published: August 2024

Background
Cellulose nanofibrils (CNFs) are advanced biomaterials that are lightweight, robust, and have low thermal expansion coefficients, making them highly attractive for diverse industrial applications. However, concerns regarding the inhalation risks require rigorous safety evaluations. This study aimed to investigate the pulmonary inflammatory effects of CNFs and their retention in the lung.
Methods
Rats were intratracheally instilled with three types of CNFs (2.0 mg/kg bw): TEMPO-oxidized CNF (CNF1), mechanically fibrillated CNF from needle-bleached kraft pulp (CNF2), and ultrashort mechanically fibrillated CNF (CNF3). Additionally, two doses of lipopolysaccharide (LPS) solution were administered intratracheally to evaluate the effects of endotoxins in the CNF suspension. The inflammatory response of the lungs induced 28 days after administration was investigated. Concerns persist regarding CNF retention in the lungs owing to their colorlessness, which complicates their spectroscopic detection in lung tissues. To address this, chemical dye-stained CNF1 (sCNF1) and CNF2 (sCNF2) were instilled in rats at 2.0 mg/kg bw, and lung samples analyzed at 7-, 30-, and 90-days post-instillation.
Results
Microscopic evaluation of the bronchoalveolar lavage fluid (BALF) revealed that alveolar macrophages had phagocytized CNFs 28 days after instillation. Histopathological examinations showed distinct patterns for each CNF type. CNF1 was phagocytosed by alveolar macrophages, leading to extensive inflammatory infiltration and macrophage degeneration/necrosis. CNF2 accumulated in terminal bronchioles, causing granuloma formation without alveolar infiltration. CNF3 induced extensive alveolar inflammation and granuloma formation. BALF analysis indicated varied leukocyte profiles. CNF1 increased total leukocytes and normal macrophage levels. CNF2 elevated total protein and lactate dehydrogenase levels without significant changes in leukocyte count. CNF3 raised total leukocytes, neutrophils, and cytokine levels (MIP-1α, IL-1β, IL-18). LPS did not induce significant changes in histopathological findings or BALF analysis, suggesting that the endotoxin contaminant in the CNF suspension had minimal effects on lung tissue. Studies with sCNF1 and sCNF2 showed that CNFs persisted in lung tissue up to 90 days post-instillation with little clearance.
Conclusions
This study highlights the significant influence of CNF properties, especially fiber diameter and length distribution due to the manufacturing method, on acute lung inflammation patterns. Additionally, inhaled CNFs were retained in lung tissue, and their clearance was not found to be easy. These results emphasize the importance of evaluating the inhalation toxicity, bioaccumulation, and clearance kinetics of CNF with different properties for safe handling and commercialization. However, future animal studies are necessary to improve our understanding of the potential toxic effects and mechanisms of CNF with different properties.