Stabilization of Ostwald ripening in low molecular weight amino lipid nanoparticles for systemic delivery of siRNA therapeutics.

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.

Assessing the heterogeneity level in lipid nanoparticles for siRNA delivery: size-based separation, compositional heterogeneity, and impact on bioperformance.

A primary consideration when developing lipid nanoparticle (LNP) based small interfering RNA (siRNA) therapeutics is formulation polydispersity or heterogeneity. The level of heterogeneity of physicochemical properties within a pharmaceutical batch could greatly affect the bioperformance, quality, and ability of a manufacturer to consistently control and reproduce the formulations. This article studied the heterogeneity in the size, composition, and in vitro performance of siRNA containing LNPs, by conducting preparative scale fractionation using a sephacryl S-1000 based size-exclusion chromatography (SEC) method. Eight LNPs with size in the range of 60-190 nm were first evaluated by the SEC method for size polydispersity characterization, and it was found that LNPs in the range of 60-150 nm could be well-resolved. Two LNPs (LNP A and LNP B) with similar bulk properties were fractionated, and fractions were studied in-depth for potential presence of polydispersity in size, composition, and in vitro silencing, as well as cytotoxicity. LNP A was deemed to be monodisperse following results of a semipreparative SEC fractionation that showed similar size, chemical composition, in vitro silencing activity, and cytotoxicity across the fractions. Therefore, LNP A represents a relatively homogeneous formulation and offers less of a challenge in its pharmaceutical development. In contrast, LNP B fractions were shown to be significantly more polydisperse in size distribution. Interestingly, LNP B SEC fractions also exhibited profound compositional variations (e.g., 5 fold difference in N/P ratio and 3 fold difference in lipid composition) along with up to 40 fold differences in the in vitro silencing activity. The impact of LNP size and formulation composition on in vitro performance is also discussed. The present results demonstrate the complexity and potential for presence of heterogeneity in LNP-based siRNA drug products. This underscores the need for tools that yield a detailed characterization of LNP formulations. This capability in tandem with the pursuit of improved formulation and process design can lead to more facile development of LNP-based siRNA pharmaceuticals of higher quality.