Magnetothermal Embolic Microspheres Triggering Ferritinophagy for Transarterial Immuno-embolization

Purpose: We aim to develop a novel immuno-embolization strategy using artesunate (ART)-loaded embolic microspheres (AMS) that trigger magnetothermal ferritinophagy to promote HCC regression.

Materials and Methods: AMS incorporating Fe₃O₄ nanoparticles was fabricated via oil-to-water emulsion. ART, Fe₃O₄, and PLGA were dissolved in DCM and emulsified in 2% PVA and homogenized. Magnetothermal stimulation triggered controlled ART release, quantified by LC-MS. In vitro ferritinophagy in HepG2 cells (human HCC, ATCC) was evaluated using CCK-8, immunostaining for NCOA4, ferritin, Fe²⁺ probes, GPx tests, and western blotting of GPX4/xCT. Immunogenic modulation was assessed through macrophage polarization markers (F4/80, CD86, CD206). In vivo, N1S1 orthotopic HCC rat models (SD rat, male, 8-10 weeks) were treated with control, AMS embolization, and AMS plus magnetothermal exposure (n = 4/group). Tumor regression was monitored via 7T MRI, and immune cells were assessed by multi-parameter flow cytometry.

Results: AMS had an average diameter of 22.9 μm, with ART loading 4.6% (efficiency 43.6%) and Fe₃O₄ loading 9.9% (efficiency 97.0%). Under magnetothermal conditions, the AMS suspension reached 48^oC (ΔT = 26^oC). Spontaneous ART release was 32% for 24 h, which increased to 70% within 20 min with magnetothermal triggering. The initial burst release of ART can induce immunogenic cell death, but prolonged exposure may induce a tolerogenic effect and limit cytotoxicity. This limitation was overcome by magnetothermal-triggered ART release, which enhanced tumor cell killing (61%) compared to AMS alone (28%). ART modulated intracellular iron metabolism by activating NCOA4, disrupting ferritin, which increased ROS and inactivated GPX. xCT inhibition further intensified oxidative stress, with magnetothermal AMS producing stronger effects than AMS alone. Consequently, both Fe²⁺ and ROS favored M1 macrophage polarization, confirmed by a 2-fold increase in the M1/M2 ratio. Lastly, in vivo, AMS plus magnetothermal exposure significantly suppressed tumor growth (8.6 mm³) compared to AMS alone (265 mm³) or control (1091 mm³). Tumor-infiltrating immune cells exhibited enhanced anti-tumor phenotypes, including mature DC (11% vs 16%), CD8⁺ T cells (9% vs 13%), IFNγ ⁺CD4⁺ T cells (2% vs 5%), and M1 macrophage (1% vs 6%).

Conclusion: AMS-mediated hepatic arterial immuno-embolization effectively induces ferritinophagy and overcomes Fe²⁺-induced immune tolerance through magneto-thermal triggered ART release, offering a promising strategy for HCC therapy.