Lipid nanoparticles (LNPs) are the most successful non-viral delivery system that have entered the clinic, which contains an ionisable lipid, a phospholipid, cholesterol and a PEGylated lipid. pKa pf the ionisable lipid is a critical property that interrelated and effect the morphology of LNPs and the way they interact with target cells. However, why biological activities differed when the pKa of the ionisable lipids in the LNPs are different is unknown.
Therefore, we selected a group of ionisable lipids, DLinMC2DMA (MC2), DLinMC3DMA (MC3) and DLinMC4DMA (MC4) that have similar chemical structures but with different pKa as a tool to determine how pKa of the ionisable lipids affects the biological activities of the LNPs. From the literature, MC2, MC3 and MC4 have the pKa around 5.8, 6.4 and 6.9, with addition of the commonly used lipids, ALC-0315 (pKa= 6.09), SM-102 (pKa = 6.7), and DODAM (pKa around 7). Then we performed quantitative SNAPswitch and SLEEQ assays and Nanoluc and mScarlet reporter gene expression to indicate the cellular uptake and LNP binding to the cells, endosomal escape and transfection efficiency.
From the Nanoluc and mScarlet expression data verses TNS pKa measurements, the level of gene expression is proportional to the increase of pKa. However, LNPs with higher pKa showed higher levels of cytotoxicity. As expected, there was a correlation between LNP association with cells and protein expression, however when taking into account the relative association, MC4 LNPs showed higher levels of protein expression. To investigate this further, we measured the amount of mRNA delivered to the cytosol. The efficiency of cytosolic delivery (endosomal escape) was lower for MC2 (~3%) compared to MC3 and MC4 (~10%). All these results suggested pKa of the ionizable lipids plays a major role in cellular association, endosomal escape and transfection.