Skip to main content

Investigation of Lipid Nanoparticle Internal Structure and Its Effect on Epithelial Cell Transfection


EMSL Project ID
51304

Abstract

Lipid-based nanoparticles (LNPs) are a highly efficient way to encapsulate and safely transport nucleic acids past the cellular wall. In the last decade, nucleic acid therapeutics have expanded towards the realm of ribonucleic acids (RNA), enabling expression of a predetermined protein while eliminating the need to pass the nuclear barrier and reducing the immune response. However, the delivery of RNA into the cells is restricted by low endosomal escape, limiting nucleic acid delivery to the cytosol. Overcoming this challenge has been addressed by modulating the lipid composition which influences the surface properties and morphology of LNPs; still, little is yet understood about the LNP design criteria to improve endosomal escape. Cryo-electron microscopy is a powerful tool to investigate the internal structure of soft nanoparticles and has a track record in revealing structural features for LNP predecessors, liposomes. The previous results established in Sahay lab with the help of PNCC confirmed that changes to LNP formulation, namely the introduction of cholesterol analogs, have distinct consequences on nanoparticle shape, lamellarity, and phase separation, which resulted in up to 200-fold improvement in gene delivery both in vitro and in vivo. These results emphasize the significance of the proposed study, suggesting that the internal structure of LNPs has tremendous implications on gene delivery efficiency. This study aims to further investigate the relationship between nanoparticle morphology and the efficiency of endosomal escape. We propose to investigate several formulation vectors that might affect internal structure: 1) modulation of PEG-lipid quantity, responsible for minimizing opsonization and improving cellular entry; 2) modulation of structural lipids (cholesterol, sitosterol, and phospholipid DSPC), providing lipid bilayer stability/rigidity and affecting interactions with endosomal wall; and 3) exposure of LNPs with varying fraction of PEG-lipid to sheer forces, which would provide insights into the potential of gene delivery via LNP-based aerosols. As a result, we intend to shed light on the structural criteria to overcome delivery limitations, thus helping to establish LNP design guidelines for next-generation RNA therapeutics.

Project Details

Start Date
2020-03-15
End Date
2021-03-17
Status
Closed

Team

Principal Investigator

Gaurav Sahay
Institution
Oregon State University

Team Members

Yulia Eygeris
Institution
Oregon State University

Theo Humphreys
Institution
Oregon Health & Science University