This study investigates how liquid crystalline inverted lipid phases in lipid nanoparticles (LNPs) enhance the delivery of small interfering RNA (siRNA) into cells. LNPs are advanced carriers for delivering RNA molecules, as demonstrated by their use in vaccines against SARS-CoV-2. The research uses a bottom-up rational design approach to create LNPs with programmable lipid phases that encapsulate siRNA. Cryogenic transmission electron microscopy (cryoTEM), cryogenic electron tomography (cryoET), and small-angle X-ray scattering (SAXS) reveal that these LNPs can form inverse hexagonal structures, which are liquid crystalline in nature. Compared to lamellar LNPs, these inverted hexagonal structures enhance intracellular silencing efficiency. The study shows that lamellar LNPs undergo a transition from lamellar to inverse hexagonal phases upon interaction with anionic membranes, while LNPs with pre-programmed inverted hexagonal phases bypass this transition, leading to more efficient one-step delivery. This rational design of LNPs with defined lipid structures improves the understanding of the nano-bio interface and enhances LNP design and use. The results indicate that liquid crystalline inverted hexagonal phases in LNPs are more thermally stable and lead to higher transfection efficiency. The study also demonstrates that these structures remain stable upon interaction with large unilamellar vesicles (LUVs), which mimic endosomal membranes. The findings suggest that pre-programmed lipid phases in LNPs can enhance RNA delivery and endosomal escape, which is a major challenge in RNA therapy. The study highlights the importance of lipid composition, RNA content, and temperature in the formation and stability of lipid structures in LNPs. The results provide insights into the mechanisms underlying LNP-membrane interactions and the efficiency of RNA delivery. The study concludes that the rational design of LNPs with defined lipid structures can improve the efficiency of RNA therapeutics.This study investigates how liquid crystalline inverted lipid phases in lipid nanoparticles (LNPs) enhance the delivery of small interfering RNA (siRNA) into cells. LNPs are advanced carriers for delivering RNA molecules, as demonstrated by their use in vaccines against SARS-CoV-2. The research uses a bottom-up rational design approach to create LNPs with programmable lipid phases that encapsulate siRNA. Cryogenic transmission electron microscopy (cryoTEM), cryogenic electron tomography (cryoET), and small-angle X-ray scattering (SAXS) reveal that these LNPs can form inverse hexagonal structures, which are liquid crystalline in nature. Compared to lamellar LNPs, these inverted hexagonal structures enhance intracellular silencing efficiency. The study shows that lamellar LNPs undergo a transition from lamellar to inverse hexagonal phases upon interaction with anionic membranes, while LNPs with pre-programmed inverted hexagonal phases bypass this transition, leading to more efficient one-step delivery. This rational design of LNPs with defined lipid structures improves the understanding of the nano-bio interface and enhances LNP design and use. The results indicate that liquid crystalline inverted hexagonal phases in LNPs are more thermally stable and lead to higher transfection efficiency. The study also demonstrates that these structures remain stable upon interaction with large unilamellar vesicles (LUVs), which mimic endosomal membranes. The findings suggest that pre-programmed lipid phases in LNPs can enhance RNA delivery and endosomal escape, which is a major challenge in RNA therapy. The study highlights the importance of lipid composition, RNA content, and temperature in the formation and stability of lipid structures in LNPs. The results provide insights into the mechanisms underlying LNP-membrane interactions and the efficiency of RNA delivery. The study concludes that the rational design of LNPs with defined lipid structures can improve the efficiency of RNA therapeutics.