Fig. 5: DLin-KC2-DMA FLuc mRNA LNPs with NP lipid:mRNA ratio in the range 2–8 have NP lipid:mRNA ratio dependent size, mRNA copy number, encapsulation efficiency, pKa, and in vitro transfection efficiency.

FLuc mRNA KC2 LNPs assembled with NP lipid:mRNA ratio 2–8 (mole ratio of the ionizable lipid KC2 amine to the mRNA phosphate) were produced with lipid mole ratios 50:10:38.5:1.5 (KC2:DSPC:Cholesterol:PEG-DMG2000). a LNP diameter (number average DLS diameter) and volume decreased with increasing NP. b Encapsulation Efficiency increased at higher NP. Encapsulated NP lipid:mRNA ratio in the LNP (NP ÷ Encapsulation Efficiency) was double the formulation NP lipid:mRNA ratio at low N:P 2 (~5) and only ~25% higher (10 vs 8) at high NP lipid:mRNA ratio 8. c The average number of mRNA copies and of each of the 5 lipids in the LNP was calculated based on the molecular volume model described in Supplementary Information. The number of mRNA copies is proportional to volume of the LNP and inversely proportional to the Encapsulated NP lipid:mRNA ratio resulting in one copy per LNP at NP 8 and 6 copies at NP 2. d TNS binding assay pKa were found by fitting TNS fluorescence data to the HH equation (lines). The TNS binding assay only detects initial LNP protonation at higher pH > 6. e The LNP zeta potential (ZP) measured over the same pH range 4–10 was shifted upwards as NP increased. Unlike the TNS binding assay, ZP detects protonation continually down to pH 4 revealing ionizable lipid protonation in the LNP at pH values corresponding to intracellular endosomes and lysosomes. pKa from ZP was found by fitting to the extended HH model. pI was found by interpolating ZP to zero potential. f pKa from TNS and ZP did not change significantly with NP lipid:mRNA ratio however pI from ZP strongly increased with NP lipid:mRNA ratio by charging the LNP positively. g LNP net charge was calculated from ZP and the DLS number size using an electrokinetic model described in Supplementary Information. h An estimate of the LNP dielectric constant was obtained by calculating a theoretical LNP charge at high pH equal to one negative charge per nucleotide (see Supplementary Information) and dividing by the charge (g) measured at high pH determined from ZP. Resulting LNP dielectric constants ranged from 6 to 24, consistent with a lipid phase (dielectric constant 5⁴⁰) containing some water³⁰ (dielectric constant 80). i Low NP LNPs exhibited a more linear dose response and were more potent at the higher doses than higher NP LNPs. j A slight reduction in cell viability measured with Alamar Blue was observed at the higher dose 200 ng but with no dependence on NP. k Cell morphology versus dose and NP was consistent with Alamar Blue in j showing some loss of viability at nigh 200 ng dose. Scale bar is 100 μm. Mean ± SD (N = 3) for i, j.