Fig. 3: In vitro transfection efficiency, toxicity, size, and encapsulation efficiency of FLuc mRNA LNPs at NP lipid:mRNA ratio 4 prepared with five commercially available ionizable lipids DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DODMA, and DODAP.

a Transfection Efficiency by luciferase assay showed LNPs were able to deliver and express FLuc provided the TNS binding assay pKa was in the range 6.5–7, corresponding to zeta potential pKa of 5.7–6.2 while the aqueous soluble and theoretical pKa’s were in the range of 8.6–9.4. Theoretical design of effective ionizable lipids could be based on having theoretical pKa’s that are 2–3 points higher than desired LNP pKa’s, in order to examine a wide design space of potential candidates. *p < 0.0001 using a multivariate (dose and LNP predicting FLuc) analyses. All pairwise comparisons were significant; we only indicate KC2 vs MC3 for clarity b Number-weighted average diameter (bars) and PDI (circles) of LNPs by DLS. c Cell Viability after transfection with LNPs with Alamar Blue. d Encapsulation Efficiency (bars) using the ribogreen assay and calculated number of mRNA copies (circles) in the LNP using the molecular volume model described in Supplementary Information. e Phase contrast cell morphology after 24 h transfection period with LNPs at different doses was consistent with Alamar Blue in C. Doses of 100 and 200 ng showed areas of cell loss (*) in DODAP, DODMA, MC3, DLin, and KC2 at these higher concentrations. Scale bar is 100 μm. Mean ± SD (N = 3).