Galactose-modified, glycyrrhizic acid-based liposomes for targeted co-delivery of bufalin: A “drug-in-as-excipient” strategy for hepatocellular carcinoma therapy

Glycyrrhizic acid (GL) entirely substitutes cholesterol as a bioactive liposomal membrane stabilizer, embodying a “drug-in-as-excipient” design. • A Quality-by-Design (QbD) approach with Box-Behnken optimization ensures formulation robustness and high bufalin entrapment efficiency (88.83%). • Dual targeting via GL-receptor and galactose-ASGPR interactions quantitatively verified by tumor targeting index (TTI) and AUTC metrics. • Gal-BF-GL-Lps achieves a 1.8-fold reduction in IC 50 , potent ROS induction, and mitochondrial apoptosis in H22 hepatoma cells. • Gal-BF-GL-Lps yields a 72.76% tumor volume inhibition rate and favorable biocompatibility in an orthotopic hepatocellular carcinoma model. HCC remains a major therapeutic challenge, and the clinical application of BF is severely limited by its poor solubility, narrow therapeutic window, and systemic toxicity. To address these obstacles, we designed a galactose-modified (Gal), glycyrrhizic acid (GL)-based liposomal system (Gal-BF-GL-Lps) that embodies a “drug-in-as-excipient” philosophy. In this design, the amphiphilic GL was introduced to replace cholesterol entirely, simultaneously functioning as a membrane stabilizer, a therapeutic synergist, and an active ligand for the glycyrrhetinic acid receptor overexpressed on hepatoma cells. The formulation was systematically optimized using a Box-Behnken design within a Quality-by-Design framework, yielding a robust preparation with an entrapment efficiency of 88.83% and a mean particle size of 131.67 nm. The surface decoration with a galactose modified ligand (Gal) further established a dual-targeting mechanism by engaging the asialoglycoprotein receptor. In vitro , Gal-BF-GL-Lps dramatically enhanced cellular uptake by H22 hepatoma cells, potentiating intracellular reactive oxygen species generation and mitochondrial membrane depolarization, which culminated in a superior apoptotic rate and a 1.8-fold lower IC 50 compared to free BF. In vivo real-time fluorescence imaging and the quantitative analysis of the tumor targeting index (TTI) and the area under the TTI-time curve (AUTC) verified the exceptional tumor-homing capacity and prolonged retention of Gal-BF-GL-Lps. This precise targeting translated into powerful antitumor efficacy in an orthotopic HCC mouse model, achieving a tumor volume inhibition rate of 72.76% and significantly downregulating key serum biomarkers (AFP, GPC-3, DCP) and inflammatory cytokines, while concurrently alleviating tumor-induced immunosuppression. Crucially, systematic biocompatibility assessments confirmed the excellent safety profile of Gal-BF-GL-Lps. This integrated, QbD-driven nanoplatform leverages the bioactive carrier material itself to achieve dual-targeted delivery and synergistic therapy, offering a highly translatable strategy for HCC treatment. Download: Download high-res image (333KB) Download: Download full-size image