The clinical effectiveness of doxorubicin (DOX) in hepatocellular carcinoma (HCC) remains constrained by poor tumor selectivity and the emergence of multidrug resistance. In this work, we examine how the electronic nature of the C-5 substituent on uracil-based nucleoside lipids shapes the physicochemical and biological behavior of DOX-loaded lipid nanoparticles (LNPs). Four ionizable lipids were designed, combining either thymidine (methyl, electron-donating) or 5-fluorouridine (FURD; fluorine, electron-withdrawing) head groups with α-tocopherol or cholesterol tails through a triazole linker, and synthesized via copper-catalyzed azide-alkyne cycloaddition, followed by formulation into DOX-LNPs. Notably, introducing the electron-withdrawing fluorine in FURD-based LNPs led to a clear improvement in DOX encapsulation efficiency (72-76% compared to 52-56%), enhanced pH-responsive drug release, and increased intracellular reactive oxygen species generation in HepG-2, Huh-7, and SK-Hep-1 cells. This redox perturbation triggers mitochondrial membrane depolarization, elevates Bax/Bcl-2 ratios, enhances cytochrome c release and caspase-3 cleavage, and induces pronounced S-phase arrest and apoptosis. Confocal microscopy further revealed markedly enhanced nuclear accumulation of DOX in the case of FURD-LNPs, while in vivo biodistribution studies showed clear preferential uptake in the liver. Density functional theory (DFT) calculations revealed distinct modulation of the frontier molecular orbital energy gap (EHOMO-LUMO) between the substituents, providing a theoretical electronic rationale that strongly correlates with these empirical trends. Taken together, these findings suggest an important correlation between head-group inductive modulation and redox-driven anticancer activity, offering a promising molecular design strategy for nucleoside-lipid nanocarriers in HCC therapy.
