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Gene therapy holds great promise for treating prostate cancer unresponsive to conventional therapies. However, the lack of delivery systems that can transport therapeutic DNA and drugs while targeting tumors without harming healthy tissues presents a significant challenge. This study aimed to explore the potential of novel hybrid lipid nanoparticles, composed of biocompatible zein and conjugated to the cancer-targeting ligand transferrin. These nanoparticles were designed to entrap the anti-cancer drug docetaxel and carry plasmid DNA, with the objective of improving the delivery of therapeutic payloads to prostate cancer cells, thereby enhancing their anti-proliferative efficacy and gene expression levels. These transferrin-bearing, zein-based hybrid lipid nanoparticles efficiently entrapped docetaxel, leading to increased uptake by PC-3 and LNCaP cancer cells and significantly enhancing anti-proliferative efficacy at docetaxel concentrations exceeding 1 µg/mL. Furthermore, they demonstrated proficient DNA condensation, exceeding 80% at polymer–DNA weight ratios of 1500:1 and 2000:1. This resulted in increased gene expression across all tested cell lines, with the highest transfection levels up to 11-fold higher than those observed with controls, in LNCaP cells. These novel transferrin-bearing, zein-based hybrid lipid nanoparticles therefore exhibit promising potential as drug and gene delivery systems for prostate cancer therapy. [ABSTRACT FROM AUTHOR]