Stabilization of Ostwald Ripening in Low Molecular Weight Amino Lipid Nanoparticles for Systemic Delivery of siRNA Therapeutics

Abstract

Lipid nanoparticles (LNPs) represent the most clinically advanced technology for the systemic delivery of therapeutic siRNA in vivo. Toward this end, a novel class of LNPs comprising low molecular weight (MW) ionizable amino lipids having asymmetric architecture was recently reported.1 LNPs of these amino lipids, termed asymmetric LNPs, were shown to be highly efficacious and well tolerated in vivo; advances were enabled by improved endosomal escape, coupled with enhanced amino lipid metabolism and clearance. In this work, we show that, in contrast to their desirable pharmacological performance, asymmetric LNPs present a significant pharmaceutical developability challenge, namely physical instability limiting extended shelf life. Using orthogonal characterization methods, we identify the mechanism of LNP instability as Ostwald ripening and establish it to be driven predominantly by the asymmetric amino lipid component. Through rational optimization of LNP physical and macromolecular properties, we are able to significantly attenuate or entirely eliminate the Ostwald ripening instability. Modulation of LNP size, for example, effectively halts particle growth. Similarly, optimization of LNP macromolecular packing through deliberate selection of structurally matched colipids significantly diminishes the rate of ripening. This later experimental observation is substantiated by molecular dynamics simulations of LNP self-assembly, which establish a quantitative dependence of LNP macromolecular order on colipid structure. In totality, the experimental and molecular dynamics outcomes of this work support the rational design of LNP physical and chemical properties leading to effective Ostwald ripening stabilization and enable the advance of asymmetric LNPs as a clinic-ready platform for siRNA therapeutics.

Direct Pharmacological Inhibition of β-Catenin by RNA Interference in Tumors of Diverse Origin

“…EnCore A-C contains DL-036, which contains a dimethylglycine head group and two symmetrical acyl chains (Figure 2A). It has been previously suggested that incorporating asymmetric acyl chains into amino lipids can facilitate pH-dependent lipid phase transitions, thereby increasing endosomal release of the payload (38–40). In a test set of otherwise identical LNPs, changing the lengths or the number of double bonds of one or both of the acyl chains significantly impacted the CTNNB1 mRNA silencing activity in tumors (Figure 2A).…”

Co-Administration of Lipid Nanoparticles and Sub-Unit Vaccine Antigens Is Required for Increase in Antigen-Specific Immune Responses in Mice

“…Lipid Nanoparticles were prepared at Merck (West Point, PA, USA) and characterized as previously described [17,20]. The LNPs are comprised of an ionizable amino lipid ((13Z,16Z)- N , N -dimethyl-3-nonyldocosa-13,16-dien-1-amine), distearoylphosphatidylcholine (DSPC), cholesterol, and poly(ethylene glycol)2000-dimyristoylglycerol (PEG2000-DMG), in a molar ratio of 58:30:10:2, respectively.…”

Modified mRNA/lipid nanoparticle-based vaccines expressing respiratory syncytial virus F protein variants are immunogenic and protective in rodent models of RSV infection

“…For Merck formulations, LNP encapsulating mRNA were prepared by rapid precipitation process as previously described. 53 The lipid components of the LNP comprised an asymmetric ionizable amino lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and poly(ethylene glycol)2000dimyristoylglycerol (PEG2000-DMG) in a molar ratio of 58:30:10:2, respectively. For formulations carried out by Moderna, LNPs were generated as described in, 34 and lipids were dissolved in ethanol at molar ratios of 50:10:38.5:1.5 (ionizable lipid: DSPC: cholesterol: PEG-lipid).…”