Mechanism of Macromolecular Structure Evolution in Self-Assembled Lipid Nanoparticles for siRNA Delivery

Abstract

Lipid nanoparticles (LNPs) are a leading platform for therapeutic delivery of small interfering RNAs (siRNAs). Optimization of LNPs as therapeutic products is enabled by the development of structure-activity relationships (SAR) linking LNP physiochemical and macromolecular properties to bioperformance. Methods by which LNP properties can be rationally manipulated are thus critical enablers of this fundamental knowledge build. In this work, we present a mechanistic study of LNP self-assembly via a rapid antisolvent precipitation process and identify critical physiochemical and kinetic parameters governing the evolution of LNP three-dimensional macromolecular structure as a biorelevant SAR feature. Using small-angle X-ray scattering, LNPs are shown to undergo a temporal evolution in macromolecular structure during self-assembly, rearranging from initially disordered phases after precipitation into well-ordered structures following a necessary annealing stage of the assembly sequence. The ability of LNPs to undergo structural reorganization is shown to be effected by the chemical nature of the aqueous antisolvent used for precipitation. Antisolvents of varying buffering species differentially influence LNP macromolecular features, revealing a new participatory role of buffer ions in LNP self-assembly. Furthermore, the formation of macromolecular structure in LNPs is shown to improve the efficiency of siRNA encapsulation, thereby offering a simple, nonchemical route for preparation of high-payload LNPs that minimize the dose of lipid excipients. The developed LNP precipitation process and mechanistic understanding of self-assembly are shown to be generalizable, enabling the production of LNPs with a tunable range of macromolecular features, as evidenced by the cubic, hexagonal, and oligo-lamellar phase LNPs exemplarily generated.

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA

“…To further investigate the internal structure of LNPs, we carried out small-angle X-ray scattering (SAXS) experiments, which have been commonly employed to study the organization of nucleic acids encapsulated in polymeric or lipidic systems [42][43][44][45] . Figure 3b and Supplementary Table 1, respectively, shows the SAXS profiles and data collection parameters of MC3 and lipid 9 nanoparticles.…”

A Tetravalent Sub-unit Dengue Vaccine Formulated with Ionizable Cationic Lipid Nanoparticle induces Significant Immune Responses in Rodents and Non-Human Primates

“…As described in our previous publications 32 33 , the lipid nanoparticle is composed of four main components: Merck proprietary ionizable cationic lipids, 1,2-distearoyl-glycero-3-phosphocholine (DSPC) phospholipid, cholesterol, and a PEGylated lipid, PEG (2000)-DMG. To specifically evaluate if reduction in the charge of the lipid nanoparticle would impact its ability to boost antigen specific immune responses, we manufactured LNPs, termed Reduced Cationic Lipid LNPs, (RCLs).…”

Encapsulation state of messenger RNA inside lipid nanoparticles

“…It comprises several lipid components, including an ionizable lipid that plays a central role in delivery efficacy (7)(8)(9). Entrapment of RNA is achieved by combining RNA with lipids at an acidic pH (10) at which the ionizable lipid is positively charged, thus ensuring a charge-driven interaction with the negatively charged nucleic acid. After a period of maturation, pH adjustment above the pKa of the ionizable lipid results in a near-neutral surface charge desirable for clinical administration (10).…”