Nanoscale Structural Insights into Human Pulmonary Mucus and Its Snail Slime-Based Surrogate Using Automated TEM...

Pulmonary drug delivery is a promising approach for treating respiratory diseases, but the mucus barrier in bronchial airways often hinders therapeutic efficacy. The nanoscale structure of mucus is critical for drug penetration, yet remains poorly understood. In this study, we quantitatively characterize the structure of patient-derived pulmonary mucus and evaluate cross-linked snail slime as a structurally relevant biomimetic model. Using high-pressure freezing, cryosubstitution, and transmission electron microscopy with automated image analysis, we reveal that pulmonary mucus exhibits a fine, interconnected fibrous network with pore sizes below 500 nm, challenging prior reports of micrometer-scale pores. Applying the same methodology to cross-linked snail slime, we demonstrate that it faithfully replicates the nanoscale architecture of pulmonary mucus, beyond its rheological similarity. This work clarifies the structural organization of mucus and establishes cross-linked snail slime as a robust model for studying drug diffusion. Our detailed protocol also provides a reproducible framework for future investigations.