Dual itaconate delivery systems modulate macrophage Acod1-Hif-1α-glycolysis axis for immunotherapy of bioprosthetic heart valve calcification

Calcification remains a major barrier to the long-term durability of bioprosthetic heart valves (BHVs), yet effective therapeutic strategies are still lacking. Emerging evidence suggests that targeting the immune response holds strong promise for mitigating BHV calcification, although the precise mechanisms remain elusive. Here, we integrated single-cell RNA sequencing, spatial transcriptomics, and multiple experimental models to elucidate the immunological mechanisms of BHV calcification and to develop targeted immunomodulatory strategies for anti-calcification therapy. The first spatiotemporal cell atlas of BHV calcification highlights macrophages as key immune drivers, confirmed by various immunodeficient mouse models. Notably, we identified a novel pro-calcification macrophage subset characterized by low Acod1 expression and reduced itaconate production. In macrophage-specific Acod1 knockout models, increased apoptosis, oxidative stress, and extracellular matrix disruption via the HIF-1α-glycolysis pathway accelerated calcification, which was reversed by itaconate supplementation. Guided by these findings, we designed two biomaterial-based therapeutic strategies: a BHV surface functionalized with itaconate via layer-by-layer assembly for localized, sustained release; and tetrazine-functionalized nanoparticles encapsulating itaconate, selectively delivered to trans-cyclooctene-modified BHVs through a bioorthogonal click reaction. Both platforms exhibited favorable biocompatibility and effectively attenuated BHV calcification in vivo, demonstrating strong translational potential. Together, our findings underscore the immune-metabolic axis underlying BHV calcification and pave the way for advanced immune-modulating treatments in BHV management.