Lipid nanoparticles (LNPs) have enabled the vaccine and therapeutic use of RNA molecules. To fully exploit the wide therapeutic potential of these types of therapies, a better understanding of the physicochemical properties-activity relationship for LNPs carrying RNA-based therapies is needed. The apparent pKa (pKa) of the instrumental ionizable lipids (ILs) in siRNA- and mRNA-loaded LNPs is one of the few physicochemical parameters that have been identified as critical for high in vivo potency. Numerous data have shown that siRNA- and mRNA-LNPs are typically potent within a narrow pKarange of ILs - pKavalues derived from the TNS assay, which likely, for the most part, probes ILs at the LNP surface. Interestingly, these data have also revealed a broad spectrum of potencies across ILs within this optimal range. Here, we discuss the pKaconcept, including factors influencing this parameter, such as co-lipids and nucleic acid molecules surrounding the ILs; why it is a critical parameter; how to measure the pKaof ILs located differently in LNPs and potential complications associated with this; and the recent use of computational methods to determine the pKavalue of ILs in LNPs. Based on the environmental impact on the pKaof ILs, it seems likely that ILs located in the LNP surface and core, either bound or unbound to mRNA, exhibit different pKavalues (or at least exhibit different propensities to be protonated). We speculate whether the internal dynamic and structural packing of the ILs sensitive to pH may also be critical to facilitate efficient endosomal escape of nucleic acid payloads. These internal features may explain some of the variability in potencies among ILs within the optimal TNS-derived pKarange. It would be rather superficial to believe that only the outside of the LNPs matters. A A A A A A A A A
