Abstract
Transcatheter arterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) is limited by inadequate intratumoral drug penetration. We developed a sequential-delivery composite gel microsphere system integrating hyaluronidase (HAase)-mediated microenvironment remodeling, glycyrrhetinic acid (GA)-mediated active targeting, and pH-responsive drug release as a potential strategy to address this barrier. Bufalin (BUF)-loaded GA-PEG-HYD-PLGA nanoparticles and HAase were co-encapsulated within carboxymethyl chitosan/sodium alginate gel microspheres containing BaSO4 for radiopacity (HAase/BUF NPs-GM). The system was characterized for particle size, drug loading, pH-responsive release, and hemocompatibility. Antitumor efficacy, deep tumor penetration, and mechanisms were evaluated in Huh-7 cells and a rabbit VX2 orthotopic liver tumor model via TACE-guided arterial delivery. HAase/BUF NPs-GM exhibited a favorable size (270 ± 61.89 μm), pH-dependent BUF release, and good biocompatibility, with an IC50 of 55.07 nM against Huh-7 cells. The system reduced hyaluronic acid content across all tumor regions, consistent with enhanced penetration of the therapeutic payload into the tumor core. Compared with controls, HAase/BUF NPs-GM combined with TACE suppressed tumor growth and angiogenesis, induced apoptosis, inhibited proliferation, and was associated with increased intratumoral CD3⁺ T cell infiltration and reduced expression of EMT-related markers. Mechanistically, the system induced ROS overproduction, mitochondrial membrane potential decrease, and G2/M cell cycle arrest. This strategy, which combines microenvironment remodeling, active targeting, and responsive release, provides a promising preclinical proof-of-concept for addressing the penetration limitations of conventional TACE. Further translational investigation, including validation in orthotopic or patient-derived xenograft models and long-term toxicity assessment, is warranted.