(E-192) The synergistic effect of ionizable lipids allows tunability of pKa in lipid nanoparticles and increased potency

Introduction:: Lipid nanoparticles (LNPs) can encapsulate nucleic acids (NA) that have therapeutic potential. The basic design of LNPs consists of four components. The main component is the ionizable lipid that makes up the supramolecular structure of the LNP. The LNP is neutral at physiological pH and becomes positively charged as the pH is lowered. The other components consist of a phospholipid to aid in stability, cholesterol to aid in lipid packing, and a PEGylated lipid to prolong the circulation time of the LNPs in vivo. The main factor known to determine LNP delivery efficiency is purported to be the pKa of the LNP, which is mainly controlled by the ionizable lipid. Herein we report that the use of two structurally different ionizable lipids in the same LNP can change the apparent pKa of the LNPs and increase transfection efficiency in vitro.

Materials and Methods:: mRNA-loaded LNPs were formulated using a total lipid concentration of 9.86 mM comprised of ionizable lipids/DSPC/cholesterol/DMG-PEG2000 in % mole ratios of 50[X:Y]/10/38.5/1.5. Controls were formulated as single ionizable lipid/DSPC/cholesterol/DMG-PEG 2000 in % mole ratios of 50/10/38.5/1.5. Each lipid was dissolved in ethanol and mixed to reach the specified molar ratios in the organic phase. mRNA encoding the firefly luciferase (FLuc) in the aqueous phase was dissolved in sodium acetate buffer (pH 4) keeping a constant lipid:mRNA ratio, or an N:P ratio (moles amine of the ionizable lipid: moles phosphate of the mRNA) of 4 prior to mixing using the Spark NanoAssmblr™. LNPs were dialyzed in sucrose buffer at pH 7.5 overnight. The LNP size and encapsulation efficiency were determined using Dynamic Light Scattering (DLS) and the Ribogreen dye exclusion assay. LNP apparent pKa and in vitro transfection efficiency (bioactivity) were determined using the 6-(p-toluidino)-2-napthalenesulfonyl chloride (TNS) binding assay and measurement of bioluminescence was carried out in HEK293 cells, respectively.

Results, Conclusions, and Discussions:: No clear pattern between size and encapsulation could be correlated with different ratios of ionizable lipids used. A combination of two ionizable lipids at specific X/Y ratios in the LNP formulations was able to increase transfection by approximately 6-fold in HEK293 cells compared to formulations containing either one of the two ionizable lipids. Certain formulations with a 7:3 ratio of ionizable lipids that include a di-ester linker were optimized for maximal transfection and are currently being tested in vivo. Our results indicate that the apparent pKa of LNPs containing two ionizable lipids is equal to the weighted average of the apparent pKa between the two ionizable lipids used in the formulations alone. In conclusion, the apparent pKa of two ionizable lipids systems can be used to fine-tune formulations by controlling the apparent pKa of LNPs. Ongoing studies are being done to determine in vitro-in vivo correlations, and why certain combinations may work better than others.


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