Folate-functionalized Zein-PEI nanoparticles for targeted cancer therapy: Integrated experimental and molecular...

The rational engineering of biopolymeric nanocarriers with enhanced biocompatibility and targetability remains a central goal in cancer nanomedicine. Here, we report the fabrication and comprehensive evaluation of zein-based nanoparticles (ZNp) surface-modified with polyethyleneimine (PEI) and folic acid-conjugated PEI (PEI-FA) for folate receptor (FR)-targeted delivery to breast cancer cells. Folic acid (FA) was covalently attached to branched PEI via EDC/NHS chemistry, with a conjugation efficiency of ∼45 %, confirmed by UV-Vis, ¹H NMR, and FTIR spectroscopy. Functionalization with PEI-FA improved colloidal stability, reduced particle size to ∼96 nm, and increased surface charge to +32 mV in physiological media. In vitro assays revealed a higher cellular uptake of ZNp/PEI-FA in FR-overexpressing MDA-MB-231 cells compared to fibroblasts, as shown by confocal microscopy. MTT analysis demonstrated reduced cytotoxicity toward normal HFF cells while preserving selective antiproliferative effects in cancer cells (IC₅₀ ≈ 52.5 μg/mL at 24 h). To gain deeper mechanistic insight, we combined experimental characterization with all-atom molecular dynamics simulations of zein-PEI and zein-PEI-FA complexes. This synergistic approach highlighted conformational adaptability and interfacial stability essential for targeting efficiency. Our integrated design framework underscores the potential of protein-based nanocarriers for safe, precise tumor delivery.