Formulation Development and In-Vitro/In-Vivo Evaluation of Dual-Drug Lipid Nanoparticle Systems for Synergistic Amelioration of Cardiac Pathology in Type 2 Diabetes Mellitus

Abstract

The study aimed to develop and evaluate co-loaded lipid nanoparticles (CL-LNPs) for synergistic management of type 2
diabetes–associated cardiomyopathy. The nanoparticles were prepared using a modified hot-emulsion ultrasonication method and
characterized for particle size, polydispersity index, zeta potential, entrapment efficiency, and drug release behavior. The
optimized CL-LNPs exhibited nanoscale dimensions with low PDI and a sufficiently negative surface charge, ensuring colloidal
stability. Entrapment efficiency for both therapeutic agents remained high, and the sustained-release profile extended up to 48
hours. Antioxidant evaluation using DPPH, ABTS, and nitric oxide assays revealed significantly higher radical scavenging
potential in CL-LNPs compared to free drugs and single-loaded formulations. Anti-inflammatory studies further demonstrated
superior inhibition of protein denaturation and enhanced stabilization of human RBC membranes. Cytotoxicity assessment on
H9c2 cardiomyocytes showed improved cell viability and reduced IC₅₀ values for CL-LNPs, while intracellular ROS
measurement and LDH leakage assays confirmed potent cytoprotective effects. Overall, the results indicate that co-loaded lipid
nanoparticles provide a promising strategy for targeting oxidative stress, inflammation, and cellular injury in diabetic
cardiomyopathy. The synergistic performance of dual-drug delivery highlights their potential for further in-vivo exploration and
therapeutic translation.