Precision RNA interference of Smad3 and Runx2 via dual targeting nanocarriers mitigates aortic valve disease.

BACKGROUND: Calcific aortic valve disease (CAVD) is a common malady with few treatment options other than valve replacement by surgery or transcatheter aortic valve implantation (TAVI). Endothelial-to-mesenchymal transition (EndMT) of valvular endothelial cells and osteogenic differentiation of valvular interstitial cells are crucial processes of CAVD. Smad3 and Runx2 are key transcription factors (TFs) that drive these processes by regulating gene expression and cellular functions. We hypothesize that downregulation of these TFs with nanoparticle-mediated RNA interference could mitigate aortic valve stenosis and calcification. METHODS: We engineered dual-targeted lipid-polymer hybrid nanocarriers (lipopolyplexes, LPP) to deliver short-hairpin RNA (shRNA) for gene silencing in pathologically remodeled aortic valve. The nanocarriers simultaneously target vascular cell adhesion molecule-1 (VCAM-1) and collagen IV, enhancing specificity toward inflamed and fibrotic valvular tissue. Encapsulated shRNA constructs were designed to silence either Smad3 or Runx2 (yielding formulations V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2). Therapeutic efficacy was evaluated in a mouse model of atherosclerosis aggravated by diabetes, mimicking the pathological environment of CAVD. RESULTS: The dual-targeted lipopolyplexes effectively facilitated gene delivery to the aortic valve, ensuring efficient transfection. Treatment with V/Cp-LPP/shSmad3 and V/Cp-LPP/shRunx2 resulted in marked silencing of Smad3 and Runx2, accompanied by significant suppression of osteogenic markers, including osteopontin, alkaline phosphatase, and osteocalcin, as well as reduced α-smooth muscle actin (αSMA) expression in valve tissue. Our data further identify Runx2 as a novel upstream modulator of Smad3 expression, unveiling a previously unrecognized Runx2-Smad3 regulatory axis with important implications for valvular pathology and targeted therapy. Beyond localized effects, systemic administration of these lipopolyplexes resulted in reduced plasma concentrations of alkaline phosphatase, cholesterol, and triglycerides, while maintaining hepatic and renal function, suggesting additional benefits for systemic metabolic homeostasis. CONCLUSIONS: These findings highlight the pivotal role of Smad3 and Runx2 downregulation in mitigating aortic valve calcification, unveiling both molecules as compelling therapeutic targets in CAVD. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12967-026-07686-1.