Optimizing Ionizable Lipid Composition to Enhance Protein Encapsulation and Delivery in Lipid Nanoparticles

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Abstract
Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four distinct formulations were generated and systematically evaluated in mammalian cell culture, leading to the identification of two lead candidates with superior delivery characteristics. The biodistribution and translocation properties of these formulations were subsequently assessed using an in vitro brain endothelial barrier model to mimic brain environment. Furthermore, we demonstrated that the selected LNPs enable efficient and functional delivery of CRISPR–Cas ribonucleoprotein complexes to mammalian cells. Together, these findings underscore the potential of rationally engineered LNPs as versatile, safe, and effective non-viral delivery platforms for advanced genome-editing applications.