Deconvoluting Lipid Nanoparticle Structure for Messenger RNA Delivery

Abstract

Lipid nanoparticle (LNP) packaged mRNA vaccines have been deployed against infectious diseases such as COVID-19, yet their structural features remain unclear. Cholesterol, a major constituent within LNPs, contributes to their morphology that influences gene delivery. Herein, we examine the structure of LNPs containing cholesterol derivatives using electron microscopy, differential scanning calorimetry, and membrane fluidity assays. LNPs formulated with C24 alkyl derivatives of cholesterol show a polymorphic shape and various degrees of multilamellarity and lipid partitioning, likely due to phase separation. The addition of methyl and ethyl groups to the C24 alkyl tail of the cholesterol backbone induces multilamellarity (>50% increase compared to cholesterol), while the addition of a double bond induces lipid partitioning (>90% increase compared to cholesterol). LNPs with multilamellar and faceted structures, as well as a lamellar lipid phase, showed higher gene transfection. Unraveling the structure of mRNA-LNPs can enable their rational design toward enhanced gene delivery.

A high-throughput Galectin-9 imaging assay for quantifying nanoparticle uptake, endosomal escape and functional RNA delivery

“…Our analysis of substitution of cholesterol to -sitosterol within LNPs confirms that particle uptake and endosomal escape can act independently of one another and highlights the importance of understanding and optimising both parameters during the design of next-generation LNPs. Cellular uptake of these particles was similar but endosomal escape was strongly improved by switching to β-Sitosterol, consistent with a recent report that -sitosterol particles have a multi-faceted structure that may directly modulate endosomal fusion 38 . Even so, little is known about if there are fundamental underlying differences in the endosomal compartments that particles are taken up in or whether there are other differences in the protein corona that forms prior to uptake that may alter particle fusion/endosomal escape properties.…”

“…Having established the mCherry-GAL9 recruitment assay and characterised its functional response to CADs, LNPs, and PNPs, we returned to investigations of how the lipid composition of LNPs influence their potency. It has been recently shown that MC3-LNPs made with -sitosterol instead of cholesterol have enhanced functional delivery of luciferase mRNA and it was suggested that this may be related to structural changes that enhance fusogenicity 12,38 . We therefore compared MC3-LNPs formulated with either cholesterol or -sitosterol and examined their uptake, ability to induce mCherry-GAL9 recruitment and functional delivery (i.e.…”

Rapid generation of circulating and mucosal decoy ACE2 using mRNA nanotherapeutics for the potential treatment of SARS-CoV-2

“…Recently, we discovered a simple substitution of cholesterol to β-sitosterol within LNP formulations boosts intracellular delivery of mRNA 22,23 . We compared the enhanced LNP formulation (eLNP: containing β-sitosterol) with the conventional LNP (containing cholesterol) as the delivery vector for hsACE2 mRNA.…”

Advances in Lipid Nanoparticles for mRNA-Based Cancer Immunotherapy

“…In this regard, the length of the alkyl tail, the flexibility of sterol ring and the polarity associated with the hydroxyl group were found essential to maintain a high transfection efficiency. Interestingly, the structural examination of lipid nanoparticles containing phytosterols revealed a polymorphic shape and various degrees of multilamellarity, polymorphism and lipid partitioning (Eygeris et al, 2020). The modification of the tail with methyl and ethyl groups led to an increase of multilamellarity (>50% increase compared to cholesterol), whereas the addition of a double bond promoted lipid partitioning (>90% increase compared to cholesterol) (Eygeris et al, 2020).…”

“…Interestingly, the structural examination of lipid nanoparticles containing phytosterols revealed a polymorphic shape and various degrees of multilamellarity, polymorphism and lipid partitioning (Eygeris et al, 2020). The modification of the tail with methyl and ethyl groups led to an increase of multilamellarity (>50% increase compared to cholesterol), whereas the addition of a double bond promoted lipid partitioning (>90% increase compared to cholesterol) (Eygeris et al, 2020). Lipid nanoparticles displaying multilamellar and polymorphic structures showed higher gene transfection.…”