Targeted YAP silencing in cardiac fibroblasts via hyaluronic acid decorated nanoparticles to prevent cardiac fibrosis

Abstract Introduction Inflammatory cells (ICs) and cardiac fibroblasts (CFs) drive myocardial fibrosis through cytokine secretion and CF differentiation into myofibroblasts, leading to excessive extracellular matrix deposition, increased stiffness, and impaired cardiomyocyte (CM) function. The YAP/TAZ transcriptional complex regulates mechanoresponsive signalling in CFs and ICs, driving their pro-fibrotic differentiation, which is prevented by Verteporfin (VTP), a specific YAP/TEAD inhibitor. Conversely, YAP is essential for CM survival after injury. Due to these opposing effects—protective in CMs but pro-fibrotic in ICs/CFs—YAP targeting is not feasible with systemic drug administration methods. Purpose To gain YAP targeting selectivity for CFs and ICs, we loaded VTP into nanoparticles (NPs) functionalized with a hyaluronic acid (HA) decay. CFs and ICs, but not CMs, express at high levels one of the main HA receptors, CD44, potentially enabling a cell-specific delivery strategy. Methods VTP loaded NPs were prepared with medical-grade materials (Chitosan and HA) by microfluidics. Human CFs were isolated from left auricles of patients undergoing aortic valve replacement and/or aortocoronary bypass, while monocytes were differentiated into M1 macrophages using pro-inflammatory cytokines. Immunofluorescence and gene expression analyses were conducted to confirm HA-NP uptake and internalization, as well as the inhibition of YAP activity. Results The internalization of NPs was monitored using rhodamine-labelled HA-NPs in CFs and ICs at 1, 2, and 4 hours. Rhodamine intensity increased over time and persisted after washout, indicating sustained intracellular retention. To demonstrate the specificity of HA-NPs uptake, silencing of CD44 by siRNA was performed. SiRNA-treated CFs showed significantly lower rhodamine uptake at 2 hours, confirming the relevance of moiety and cell-receptor interaction for NPs internalization. To assess whether the internalized HA-NPs efficiently released VTP, experiments were performed using RNA of CFs incubated with the VTP-loaded nanoparticles. Results showed a significant downregulation of YAP target genes (CTGF, CYR61 and ANKRD1), after only 2 hours of incubation in CFs, demonstrating selective and effective pathway inhibition. NP behavior was assessed in a 3D bioprinted gelatin–alginate model containing cardiac fibroblasts. Rhodamine-labelled HA-NPs showed efficient uptake within the constructs, confirming internalization in a physiologically relevant setting. Constructs exposed to VTP-loaded HA-NPs were analysed by RT-qPCR to validate the downregulation of YAP target genes also in 3D, thus confirming effective VTP delivery in a biomimetic environment. Conclusion These findings demonstrate that targeted delivery of Verteporfin via HA-NPs enables selective YAP inhibition in CFs, reducing their pro-fibrotic activation. This opens a novel therapeutic application to reduce cardiac fibrosis with a cell-targeted delivery strategy.