Oxidized pullulan-zein-caseinate hybrid nanoparticles for piperine delivery: Rational design supported by molecular...

Polysaccharides offer unique molecular scaffolding that can significantly enhance the stability and functionality of protein-based nanocarriers. Here, we demonstrate that oxidized pullulan critically stabilizes zein-caseinate nanoparticles through a combined computational and experimental strategy. Molecular dynamics simulations first revealed that pullulan promotes extensive hydrogen bonding, reduces conformational fluctuations, and compacts the protein assembly, predicting enhanced structural integrity and pH-responsive behavior. These simulations provided complementary molecular-level insights into OP-protein interactions, which, together with experimental considerations, supported the subsequent development of the nanoparticle system. Oxidized pullulan was covalently conjugated to caseinate via Schiff base chemistry and integrated into zein nanoparticles. Experimental characterization confirmed selective oxidation, efficient caseinate-oxidized pullulan conjugation, and preservation of the polymer backbone, resulting in nanoparticles with uniform size (~220 nm), narrow PDI, high redispersibility, and remarkable colloidal stability under physiological and serum-rich conditions. Piperine, a hydrophobic anticancer agent, was efficiently encapsulated, exhibiting sustained, pH-triggered release (up to ~73% at pH 5.5) consistent with the predicted polysaccharide-mediated stabilization. In vitro cytotoxicity demonstrated preferential anti-tumor activity MDA-MB-231 cells (IC₅₀ = 129 μg/mL) while sparing normal fibroblasts (IC₅₀ = 176 μg/mL). Confocal microscopy showed higher cell-associated fluorescence in MDA-MB-231 cells than in HFF cells, consistent with differential cellular association. Collectively, these findings demonstrate the potential of pullulan-functionalized protein nanoparticles to provide improved stability, redispersibility, and pH-dependent drug release, together with differential cellular association, supporting their potential as protein-polysaccharide nanocarriers for anticancer drug delivery.