Ionization Behavior of Amino Lipids for siRNA Delivery: Determination of Ionization Constants, SAR, and the Impact of Lipid pK_a on Cationic Lipid−Biomembrane Interactions

Abstract

Ionizable amino lipids are being pursued as an important class of materials for delivering small interfering RNA (siRNA) therapeutics, and research is being conducted to elucidate the structure-activity relationships (SAR) of these lipids. The pK(a) of cationic lipid headgroups is one of the critical physiochemical properties of interest due to the strong impact of lipid ionization on the assembly and performance of these lipids. This research focused on developing approaches that permit the rapid determination of the relevant pK(a) of the ionizable amino lipids. Two distinct approaches were investigated: (1) potentiometric titration of amino lipids dissolved in neutral surfactant micelles; and (2) pH-dependent partitioning of a fluorescent dye to cationic liposomes formulated from amino lipids. Using the approaches developed here, the pK(a) values of cationic lipids with distinct headgroups were measured and found to be significantly lower than calculated values. It was also found that lipid-lipid interaction has a strong impact on the pK(a) values of lipids. Lysis of model biomembranes by cationic lipids was used to evaluate the impact of lipid pK(a) on the interaction between cationic lipids and cell membranes. It was found that cationic lipid-biomembrane interaction depends strongly on lipid pK(a) and solution pH, and this interaction is much stronger when amino lipids are highly charged. The presence of an optimal pK(a) range of ionizable amino lipids for siRNA delivery was suggested based on these results. The pK(a) methods reported here can be used to support the SAR screen of cationic lipids for siRNA delivery, and the information revealed through studying the impact of pK(a) on the interaction between cationic lipids and cell membranes will contribute significantly to the design of more efficient siRNA delivery vehicles.

Degradable lipid nanoparticles with predictable in vivo siRNA delivery activity

“…Studies have shown that the ability of materials to take on a positive charge with decreasing pH may confer transfection efficacy which would point to pKa as a critical parameter 35,40 . Data in Fig.…”

Multiparametric approach for the evaluation of lipid nanoparticles for siRNA delivery

“…Of these three, the LNP pK a showed the strongest correlation with biological barriers and gene silencing. LNP pK a was measured using a well-known 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) assay (17,39) by titrating the LNPs from pH 2.5-8.5. The pK a of each fully formulated LNP was determined from the resulting fluorescence titration S-curve using a curve-fit analysis.…”

“…The pK a of each fully formulated LNP was determined from the resulting fluorescence titration S-curve using a curve-fit analysis. The advantage of this method is that LNP structure and formulation are taken into account and thus the measured pK a represents that of the LNP as a whole rather than just the individual lipid (17). The measured pK a values (Fig.…”

“…To address some of these challenges, single-parameter studies that evaluate the effect of chemical structure on a single biological property or on delivery performance have been carried out (16)(17)(18)(19)(20)(21). Furthermore, high-throughput synthetic methods have been exploited for the accelerated discovery of potent lipid nanoparticles (LNP) and evaluation of structure activity relationships (SAR) (22,23).…”

Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo**

“…In fact, a LNP formulation based on DLin-MC3-DMA (16), one of the most potent lipids identified in this work, is currently being utilized in a clinical trial for treating hypercholesterolemia; the target is PCSK9 and the data (not shown) indicate decreases in LDL-C after a single low dose. [17,18] Received: April 27, 2012 Published online: July 10,2012 . Keywords: drug delivery · gene silencing · ionizable amino lipids · liposomes · siRNA …”