Construction of 3D BEAS-2B spheroids using a chemically defined hyaluronic acid-based hybrid hydrogel for 1-nitropyrene toxicity evaluation

Abstract Air pollutants pose a severe threat to respiratory system health. Establishing an in vitro toxicological assessment model that can closely mimic the in vivo physiological state is of great significance for replacing animal experiments and improving the accuracy of toxicity prediction. Conventional two-dimensional (2D) cell culture fails to recapitulate the tissue microenvironment and pollutant concentration gradients, often resulting in deviations from the true physiological conditions in the assessment of toxicity responses. Therefore, this study was dedicated to constructing an in vitro 3D cell model suitable for the toxicity assessment of air pollutants. A thiol-cytosine dual-functional hyaluronic acid precursor (HA-SH-HMDA-Cytosine) was synthesized through chemical modification. Subsequently, a dual-network biomimetic hydrogel (Hybrid gel) was constructed through the synergistic interplay between covalent crosslinking of disulfide bonds and physical crosslinking formed by triple hydrogen bonds between cytosine and its tautomer. The obtained hydrogel possessed appropriate gelation time, porous microstructure, swelling behavior and mechanical strength. After being functional modified with RGDS peptide, the hydrogel can efficiently support the adhesion and proliferation of human bronchial epithelial BEAS-2B cells, enabling the cells to self-assembe into structurally regular lung spheroids, and its culture performance is comparable to that of Matrigel. Meanwhile, 1-nitropyrene (1-NP) was selected as a model air pollutant for toxicity assessment. The results demonstrated that the toxicity response of this lung spheroid model was more physiologically relevant than that of traditional 2D culture, accompanied by a significantly elevated IC₅₀ value. Mechanistic studies revealed that 1-NP induced excessive reactive oxygen species production, triggered lipid peroxidation and cell membrane damage, and down-regulated the expression of tight junction proteins including ZO-1, Occludin and Claudin-1, thereby impairing the airway epithelial barrier function. In summary, the 3D cell culture system based on HA biomimetic hydrogel developed in this study provides a novel, stable and reliable alternative model for the in vitro toxicological assessment of air pollutants, and offers a feasible strategy for the reduction, replacement and refinement of experimental animals in accordance with the 3R principles.