Mucus is responsible for controlling transport and barrier function in biological systems, and its properties can be significantly affected by compositional and environmental changes. In this study, the impacts of pH and cacl 2 were examined on the solution-to-gel transition of mucin, the primary structural component of mucus. Microscale structural changes were correlated with macroscale viscoelastic behavior as a function of pH and calcium addition using rheology, dynamic light scattering, zeta potential, surface tension, and ftiR spectroscopic characterization. Mucin solutions transitioned from solution to gel behavior between pH 4-5 and correspondingly displayed a more than tenfold increase in viscoelastic moduli. Addition of CaCl 2 increased the sol-gel transition pH value to ca. 6, with a twofold increase in loss moduli at low frequencies and tenfold increase in storage modulus. changing the ionic conditions-specifically [H + ] and [ca 2+ ]-modulated the sol-gel transition pH, isoelectric point, and viscoelastic properties due to reversible conformational changes with mucin forming a network structure via non-covalent cross-links between mucin chains. Mucin is a polyelectrolyte glycoprotein found in mucus, the structured complex fluid found in all types of organisms from bacteria to humans. In vertebrates, mucus is produced by mucus membranes and can be found lining the eyelids, mouth, and nose, as well as gastrointestinal, respiratory, and genital tracts. Since the 1970s 1 , animal mucus and mucin solutions have been well studied, especially the roles mucus plays in drug delivery 2-6 , disorders like cystic fibrosis 7,8 , and its protective biological functions 9-12. Mucin glycoproteins are present in mucus at concentrations of 1-5%, along with electrolytes (ca. 1%), lipids (1-2%), other proteins (1-2%), and water (90-95%) 13. Despite being present in low concentrations, mucin glycoproteins are primarily responsible for the protective and lubricative functions of mucus within the body. Solubilized mucin behaves as a complex fluid with changing viscoelastic and structural properties in response to its environmental conditions. Mucin glycoproteins are responsible for the majority of the physical properties of mucus, as mucin conformation, intra-and inter-strand bonding, and microstructure changes in response to environmental factors such as pH 6,7,12,14 , temperature 2,15 , and ion content 1,14,16. All these factors affect the reversible supramolecular bonding between functional groups within the protein strands, which modifies the microstructure as well as the mechanical and transport properties of the mucin network. In aqueous solutions, biopolymers like mucin form networks that can change dynamically due to non-covalent crosslinks, including physical entanglements and supramolecular bonding. Mucins resemble a bottle brush polymer with a protein backbone and oligosaccharide (carbohydrate) side chains arranged radially from the backbone. The protein backbone of the mucin biopolymer has areas that are dense w...

Mucus is a complex fluid that maintains moisture and simultaneously acts as a barrier and facilitates transport of select materials between the body and adjacent fluids. While mucus is comprised primarily of water, it is highly heterogeneous containing the biopolymer mucin along with lipids, salts, DNA, proteins, and cells. Complex mechanisms control the reversible network formation observed in mucus and mucin solutions. Some isolated relationships between biopolymer network structure, pH and ionic strength, and rheology have been identified; however, a complete understanding of the interplay in these mechanisms is lacking. In this effort, rheology of native mucus and mucin solutions was examined as a function of pH and salt concentration. Bulk rheology of native and artificial lung mucus confirmed that native mucus displays a solid-like behavior at low strain values. Mucus displays this solid-like gel behavior at pH values ca. 4 and below, and displays solution behavior at higher pH values. Ion concentration also plays an important role with divalent cations (Ca2+) reducing the viscosity of gelled mucin and increasing the viscosity of solution-state mucin. In addition to rheological characterization, mucin-mucin and mucin-solute interactions and resultant changes in microstructure were also studied. Morphological and chemical changes at the nano-scale were correlated to the micro-structural changes observed with rheology. Dynamic light scattering showed mucin polymer particle size heterogeneity as well as a significant increase in particle size at pH values ca. 4 and below — where the solutions display a gel behavior. Using zeta potential a decrease in dispersive forces was observed that allows for polymer-polymer interaction and particle aggregation at low pH values. Atomic force micrography showed spheroid-like aggregates between adjacent mucin particles under acidic conditions. The ability to understand and control the reversible association of network structures in polymer and biopolymer systems, such as mucin, through supramolecular interactions has fundamental impacts in the field of polymer science and engineering. There is also significant potential to advance applications involving novel hydrogel materials, such as disease treatments and drug delivery.