Optimization of DOTAP/Cholesterol Cationic Lipid Nanoparticles for Nucleic Acid Delivery

Effective delivery methods are essentiel for the successful clinical application of nucleic acids. This chapter outlines a detailed protocol for the preparation, optimization, and characterization of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (chol) lipid nanoparticles (LNPs) as efficient delivery systems for mRNA, plasmid DNA (pDNA), and oligonucleotides. We describe the formulation of LNPs using a thin-film hydration technique, optimization of key parameters (e.g., DOTAP/Chol ratios, PEGylation, and lipid-to-nucleic acid ratios), and their evaluation using physicochemical and biological assays. This protocol includes methodologies for gel retardation, particle size and zeta potential characterization, in vitro transfection assays, cytotoxicity testing, and long-term stability studies. By following this step-by-step guide, researchers can achieve reproducible results in designing LNPs for various therapeutic applications.