Transition Temperature-Guided Design of Lipid Nanoparticles for Effective mRNA Delivery

Lipid nanoparticles (LNPs) are promising mRNA delivery vehicles due to their biocompatibility and tunable characteristics. While current rational design approaches focus on ionizable lipids' p<i>K</i>_a and zeta potential to optimize mRNA encapsulation and endosomal escape, the selection of helper lipids remains largely empirical. We propose that the lipid transition temperature (<i>T</i>_m), marking the shift from the gel to the liquid crystalline phase, can guide rational helper lipid selection. Through screening 54 ionizable lipids, we identified H7T4, which displayed favorable physicochemical properties when combined with its tail variants but exhibited poor transfection efficiency. Using nano differential scanning calorimetry (nDSC) and biological small-angle X-ray scattering (BioSAXS), we found that lowering the system's <i>T</i>_m by combining H7T4 (high transition temperature) with a low-transition-temperature helper lipid such as 1,2-dioleoyl-<i>sn</i>-glycero-3-phosphoethanolamine (DOPE) significantly enhanced mRNA cellular uptake both <i>in vitro</i> and <i>in vivo</i>. These findings establish <i>T</i>_m as a crucial parameter for a rational LNP design.