Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by ¹H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties

Aug 22, 2023Molecular pharmaceutics

How Lipid Types Affect the Structure of siRNA-Carrying Nanoparticles Revealed by Molecular-Level Analysis

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Abstract

Incorporating DSPC and cholesterol in ionizable lipid-based nanoparticles decreased the molecular mobility of ionizable lipids.

DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of their hydrophobic tails.
The decreased molecular mobility and changed orientation of lipid mixtures helped maintain the stacked bilayer structure of siRNA and ionizable lipids.
This structural maintenance contributed to increased siRNA encapsulation efficiency within the lipid nanoparticles.
NMR relaxometry indicated that incorporating neutral lipids enhanced PEG chain flexibility at the interface of the nanoparticles.
A small amount of DSPC improved PEG chain flexibility, which may enhance dispersion stability and lead to a narrower size distribution of the nanoparticles.
Excess amounts of DSPC and cholesterol led to the formation of deformed particles and demixing of cholesterol within the nanoparticles.

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H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl--glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs. 1sn

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Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by ¹H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties

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