Development and characterization of a third-generation S-protected alginate-cystine thiomer

Thiomers show an improved mucoadhesion by forming disulfide bonds with mucin; however, conventional 1st-generation thiomers containing free thiol groups suffer from oxidative instability and limited shelf life. To overcome these drawbacks, this study reports the synthesis and characterization of a 3rd-generation S-protected thiomer based on alginate modified with cystine, a biogenic disulfide ligand that provides stability while retaining thiol-disulfide exchange capacity. The alginate-cystine conjugate was synthesized via carbodiimide. FT-IR and ¹³C NMR confirmed the formation of disulfide linkages between alginate and cystine, while TGA revealed increased decomposition temperatures, indicating enhanced structural stability. Quantification of thiol and disulfide content demonstrated that the S-protected thiomer contained a higher proportion of disulfide bonds and fewer free thiol groups than 1st-generation L-cysteine-modified alginate. Interaction between thiomer and porcine mucin, as an in vitro simulator of intestinal mucus, was studied as an approach of mucoadhesiveness by rheology, showing a marked shift from viscous to elastic behavior (G' > G″) with the S-protected thiomer showing >200-fold increase in G' and > 120-fold increase in complex viscosity compared to unmodified alginate. A qualitative assay was also designed to confirm ex vivo mucoadhesiveness by depositing a fluorescent derivative of the S-protected thiomer directly on fish intestine and visualizing its presence after several washes using fluorescent microscopy and spectral imaging. The thiomer also exhibited high biocompatibility through hemolysis and cytotoxicity assays. In summary, the incorporation of cystine as a low-reactivity disulfide ligand into alginate yields a S-protected thiomer with enhanced chemical stability, improved mucoadhesion and biocompatibility.