A Nanoparticle-Based Strategy to Stabilize 5-Azacytidine and Preserve DNA Demethylation Activity in Human Cardiac Fibroblasts

<b>Background:</b> 5-Azacytidine (5-Aza) is a clinically important DNMT inhibitor with the potential to modulate cardiac remodeling by epigenetically reprogramming human cardiac fibroblasts (HCFs). However, its clinical utility is limited by rapid hydrolytic degradation. Nanoparticle (NP) encapsulation offers a strategy to mitigate this instability. This study evaluated the physical and chemical stability of free 5-Aza and 5-Aza-loaded lipid nanoparticles (5-Aza-NP) under different storage temperatures and examined their effects on DNA methylation-related gene expression in HCFs. <b>Methods:</b> Hyaluronic acid-stabilized lipid NPs were prepared using a solvent displacement method. Particle size, polydispersity index (PDI), and zeta potential were monitored over four days at -20 °C, 4 °C, and 30 °C. Chemical stability was assessed using HPLC and first-order kinetic modeling. Functional activity was evaluated by treating HCFs with free 5-Aza or 5-Aza-NP stored for 96 h and measuring <i>DNMT1</i>, <i>DNMT3A</i>, and <i>DNMT3B</i> expression by RT-qPCR. <b>Results:</b> 5-Aza-NP remained physically stable at 4 °C, while -20 °C induced aggregation and 30 °C caused thermal variability. Free 5-Aza degraded rapidly at 30 °C (6.56% remaining at 72 h), whereas 5-Aza-NP preserved 11.54%. Kinetic modeling confirmed first-order degradation, with consistently longer half-lives for the NP formulation. Functionally, 5-Aza-NP preserved its ability to suppress <i>DNMT1</i> expression following 96 h of storage at 4 °C, whereas free 5-Aza showed reduced activity. In contrast, <i>DNMT3A</i> and <i>DNMT3B</i> levels remained low and unchanged across all treatments. <b>Conclusions:</b> NP encapsulation enhances the physicochemical stability of 5-Aza and preserves its <i>DNMT1</i>-inhibitory activity, while <i>DNMT3A/B</i> remain unaffected. These findings support NP-based delivery as a promising strategy to stabilize labile epigenetic drugs such as 5-Aza.