Mucus, a thick and slimy secretion produced by submucosal cells, covers many epithelial surfaces in mammalian organs and prevents foreign particles that enter the body from accessing cells. However, the mucus layer also represents a potential barrier to the efficient delivery of nano-sized drug delivery systems (polyplexes, lipoplexes, particles) to the underlying mucosal epithelium. Many studies have considered the ability of nano-sized particles and polymers to diffuse within the mucosal network using a range of different techniques, including multiple-particle tracking (MPT), diffusion chamber studies and fluorescence recovery after photobleaching (FRAP). This review highlights the current understanding of the interaction of the diffusion of nano-sized structures within mucosal networks. Moreover, this article presents an introduction to pulsed-gradient spin-echo NMR (PGSE-NMR), a potential new tool to investigate the mobility of molecular species through mucosal networks and related biological gels.