Nanoparticles across mucosal barriers: Differentiating mucoadhesion from mucopenetration using single particle tracking

Mucus is a viscoelastic, selectively permeable barrier that protects epithelial surfaces throughout the body. Its complex structure presents a major challenge for nanoparticle-based drug delivery, requiring a balance between mucoadhesion, to prolong residence time, and mucopenetration, to access underlying tissues. These processes vary by anatomical site due to differences in mucus turnover and composition. Current methods often fail to clearly distinguish between mucoadhesion and mucopenetration. Here, we highlight single-particle tracking (SPT) as a powerful approach to address this gap. By capturing the trajectories of individual nanoparticles in mucus, SPT offers high-resolution insight into their diffusion behavior and interaction dynamics. Notably, particle behavior observed in ex vivo mucus has shown good correlation with in vivo outcomes, making SPT a promising tool for translational research. Despite its advantages, SPT remains underutilized in mucosal drug delivery, largely due to a lack of standardized protocols and benchmark materials. We advocate for the development of a nanoparticle test set with well-defined surface properties (e.g., charge, hydrophobicity) and the routine reporting of key parameters such as diffusion coefficients and mean squared displacement. These steps are essential to improve reproducibility, support cross-study comparisons, and accelerate progress in mucosal nanomedicine.