Gastrointestinal (GI) mucus serves as a dynamic hydrogel barrier critical for epithelial protection, nutrient absorption, and drug delivery, yet its molecular responses to physiological and therapeutic perturbations remain underexplored. This study investigates the in vitro dynamics of mucin gels under biologically relevant conditions using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy with Two-Dimensional Correlation Spectroscopy (2DCOS). We examined mucin responses to shear (mimicking peristalsis), dilution (hydration effects), pH variations (gastric to intestinal transitions), bile salts (emulsification), exosomes (cellular signaling), and various drugs. Spectral analysis revealed sequential molecular changes, including alterations in mucin structure, conformation, hydrogen bonding, and disulfide linkages. Additionally, we explored mucin's role as a natural excipient, employing isothermal titration calorimetry to quantify binding stoichiometry, entropy, and enthalpy changes upon drug binding to mucins, while demonstrating its capacity to stabilize drug supersaturation through hydrophobic and electrostatic interactions, thereby preventing precipitation and enhancing bioavailability. These findings elucidate mucus as an adaptive biomaterial, with implications for designing mucin-inspired scaffolds in regenerative engineering, improving oral drug formulations, and modeling GI tissue responses in disease states such as inflammatory bowel disease. This in vitro work establishes much of the necessary experience needed to implement 2DCOS in vivo and advance the convergence of materials science and biology for targeted therapeutic interventions in the GI tract.
