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

Siddharth Patel,Adam A Esposito,Kevin Welsher,N Ashwanikumar,Gaurav Sahay,Tatiana Ketova,Shangguo Hou,Ema Robinson,Örn Almarsson,John L Joyal,Yan Xia,Joseph P Griffith,Cosmin Mihai

doi:10.1038/s41467-020-14527-2

Abstract

Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.

Highlights

Much success towards enhanced nucleic acid delivery has been gained through screening or designing new ionizable lipids that can perturb the endosome[12,13,14]
We identify a class of cholesterol analogs with an alkyl substitution at C-24, which, when incorporated inside an enhanced lipid-based nanoparticles (LNPs), can result in substantial increase in gene delivery
We found that cellular deficiency in Niemann Pick Type C-1 (NPC1) resulted in enhanced transfection for both, LNP and enhanced LNP (eLNP), with eLNP again demonstrating superior efficacy (Fig. 5a, b)

Summary

Introduction

Much success towards enhanced nucleic acid delivery has been gained through screening or designing new ionizable lipids that can perturb the endosome[12,13,14]. Underscoring the magnitude of cholesterol’s influence on intracellular delivery, cholesterol, and cholesterol transport proteins have been implicated to play a part in the cytosolic delivery of viruses and endocytic recycling/retention of nanoparticles[19,20,21] Drawing from this knowledge, we decode the structural characteristics of cholesterol that are crucial for efficient intracellular delivery and improved gene transfection. We identify a class of cholesterol analogs with an alkyl substitution at C-24, which, when incorporated inside an enhanced LNP (eLNP), can result in substantial increase in gene delivery These eLNPs are morphologically distinct than the traditional LNPs. The intracellular behavior of eLNPs is different, suggesting that the structural aberrations and potentially altered surface lipid composition can lead to differential trafficking. Through the use of cholesterol analogs, we can refine the nanoparticle structure, possibly arising from surface composition, leading to productive intracellular trafficking of nanoparticles, thereby enhancing cytosolic delivery

Methods

Results

Conclusion