RNA Size and Structure Modulate the Apparent pKa of Ionizable Lipid Nanoparticles

Ionizable lipid nanoparticles (LNPs) underpin today's RNA medicines by shielding nucleic acids in the bloodstream and transforming into membrane-active cationic nanoparticles inside acidifying endosomes. This behavior critically depends on the apparent pKa of LNPs, which can be tuned by altering the chemistry of lipids/lipidoids and the ratios of helper lipids. However, formulators typically assume that different RNA payloads exert identical effects on LNP ionization. In this study, we challenge this assumption and quantify how RNA modulates LNP pKa. LNP formulations with different ionizable lipidoids (SM-102, cKK-E12, ALC-0315, 306Oi10, XMAN6, and C12-200) were used to encapsulate various RNA cargos, which ranged from 21-nt to 4.5-kb mRNAs. Apparent pKa values were determined using a TNS fluorescence titration assay. RNA payload size impacted the apparent pKa of the LNPs by 0.1-0.5 pH units, the direction and magnitude of the change being dependent on the structure of the ionizable lipidoid. Moreover, same-length 21-nt siRNA and miRNA cargos produced a 0.15 pH-unit difference in XMAN6 LNPs, indicating that RNA structural features can modulate apparent pKa independently of payload size. With the monoamine lipidoid SM-102, short RNAs lowered the apparent pKa relative to mRNA-loaded particles, whereas the multiamine lipidoids cKK-E12 and XMAN6 exhibited a monotonic decrease in apparent pKa as RNA length increased, plateauing at ∼1.9 kb. The most pronounced discrepancies between apparent pKa values of lipidoid particles and corresponding LNPs (of ≤0.5 pH units) were observed with the triamine lipidoid XMAN6, pointing to a role of amino group multiplicity in the modulation of apparent pKa. These findings demonstrate that RNA payload size is an underappreciated lever for tuning LNP ionization behavior and that considering RNA payload size when conducting lipid/lipidoid library screens could enhance the development of future RNA-based therapeutics.