Intermolecular interactions modulate the assembly and structure of Gli-CMCS nanoparticles and their influence on quercetin encapsulation and digestive release.

Gliadin nanoparticles are promising carriers for hydrophobic bioactive compounds; however, their poor colloidal stability and limited control over gastrointestinal release restrict practical applications. Although gliadin-carboxymethyl chitosan (Gli-CMCS) nanoparticles have recently been developed for bioactive delivery, the molecular mechanisms by which specific intermolecular interactions contribute to nanoparticle assembly, structural evolution, and digestive release behavior remain insufficiently understood. This study aimed to develop Gli-CMCS nanoparticles as delivery carriers for quercetin and to investigate their formation mechanism, physicochemical properties, encapsulation behavior, and in vitro digestion characteristics. A combination of spectroscopic analyses, interaction force probing, and physicochemical characterization was employed to elucidate the assembly mechanism, structural evolution, stability, and delivery performance of Gli-CMCS nanoparticles. The results showed that CMCS incorporation significantly influenced nanoparticle assembly and stability. Spectroscopic and interaction force analyses revealed that gliadin interacted with CMCS through hydrogen bonding and electrostatic interactions, resulting in conformational rearrangement and nanoparticle assembly. Appropriate CMCS addition (Gli:CMCS ratios of 2:1-1:1) resulted in nanoparticles with a relatively narrow size distribution (PDI 0.30-0.37) and improved colloidal stability. Quercetin was efficiently encapsulated in the nanoparticles, achieving a maximum encapsulation efficiency of 93.92% at a quercetin concentration of 0.04 mg/mL. In vitro digestion studies demonstrated that CMCS incorporation retarded the apparent release of quercetin and improved its retention under simulated gastrointestinal conditions. These findings provide mechanistic insights into how protein-polysaccharide interactions contribute to nanoparticle assembly and are associated with digestive behavior, offering a theoretical basis for the rational design of protein-based delivery systems for hydrophobic bioactive compounds.