Genipin crosslinks purified commercial pig gastric mucin to obtain a biomimetic mucus gel

Mucins, the main constituents of mucus, play a key role in providing protection, lubrication and mediating biological signalling, transport and immune response in epithelial tissues in animals. This is a consequence of the highly complex chemical composition of the proteoglycans, and of the viscoelastic properties of mucus. Despite the high traction and advances in reproducing the material and biological properties of mucus, there is a need for robust, tuneable and reproducible methods and materials for in vitro biological assays, mucoadhesion/mucopenetration studies of pharmaceutical formulations, gut microbiome, among other. In this work, we document the development of a mucus biomimetic material based on purified commercially available pig gastric mucin (PGM), using an uncovered novel approach to covalently crosslink it using genipin, a natural agent known to crosslink chitosan and proteins. To this end, we conducted spectroscopic, biophysical and biological studies. UV/VIS spectroscopy evidence that the chemical genipin crosslinking reaction kinetics for PGM Type II and Type III had different reactivity (Ea = 108 and 37 kJ/mol, respectively) and in both cases, resulted in the formation of a dark blue pigment. By dialysis diffusion, it was demonstrated that genipin fully crosslinked PGM after one week of incubation at 37 °C. FTIR spectroscopy of the freeze-dried mucus biomaterial provided further evidence of PGM crosslinking indicating a greater curing process on Type III than Type II mucin. The blue fluid gel obtained biomaterial was found to diffuse in paper similarly to commercial ink, while DLS measurements also confirmed the increase in particle size with crosslinking time. Oscillatory rheology provided unequivocal evidence that the crosslinking process at genipin/mucin ratios ≥0.08 led to the formation of a viscoelastic gel material, with mechanical strength directly dependent on the crosslinking genipin/mucin (G/M) ratio (G‘ = ca. 10 Pa and G” = ca. 1 Pa for G/M 0.11). These gels also behaved as plastic fluid material. SEM imaging revealed that the crosslinked PGM can be used to spin coat polycarbonate porous membranes by depositing a homogeneous layer of tuneable thickness (ca. 0.6 to 6 µm). Biological in vitro assays revealed that the PGM crosslinked with genipin for up to 11 weeks was a fully cytocompatible biomaterial. Also, when used as a substrate to spin coat glass coverslips, it was shown that it can be used in bacterial biofilm assays to discern between the growth of mucin-binding (Bacteroides thetaiotaomicron and Clostridioides difficile) and non-binding (Lactobacillus paracasei) species and non-degrading ones. Altogether, our results anticipate several applications in research and as an advanced material for the developed genipin-crosslinked PGM biomimetic biomaterial. To name a few, for coating glass, plastic or porous surfaces for research studies such as gut microbiome, drug permeability, in vitro gut digestion, and 3D bioprinting.