LNP-mediated siRNA transfer: Mechanism of endosomal escape

bioRxiv (Human Technopole)

Overview

Researchers have developed a cell-free system using giant unilamellar vesicles (GUVs) to demonstrate that siRNA can cross endosomal membranes without causing membrane rupture. The study finds that acidification promotes siRNA translocation via lipid association while preserving the membrane’s barrier function. This provides a platform to optimize lipid nanoparticle (LNP) formulations for higher efficacy and lower toxicity.

In Depth

Key Findings

The study demonstrates that siRNA transfer from lipid nanoparticles (LNPs) across endosomal membrane mimics can occur without the detectable dissipation of the transmembrane pH gradient or membrane rupture. Using a cell-free system, the researchers found that acidification promotes LNP-membrane association and lipid redistribution, enabling siRNA translocation into the receiving lumen while maintaining the membrane’s barrier function.

Technical Details

To dissect the mechanism of delivery, the researchers employed giant unilamellar vesicles (GUVs) with a lipid composition mimicking early endosomes. The following technical observations were made:

• Imaging: Cryo-electron tomography revealed local interfacial lipid reorganization but no detectable membrane rupture.
• Diffusion: Fluorescence correlation spectroscopy showed heterogeneous diffusion of translocated siRNA, indicating the presence of both free and lipid-associated species.
• Complexes: Pre-formed RNA-lipid complexes exhibited pH-dependent dissociation and heterogeneous diffusion, suggesting that lipid association is a likely cause of slower luminal diffusion.
• Inhibitors: Preincubation with serum or apolipoprotein-E was found to reduce the translocation of siRNA into the GUV lumen.

Background & Context

Endosomal escape is a primary limiting factor in RNA delivery via LNPs. Previous concerns suggested that if escape requires membrane rupture, productive delivery would be intrinsically linked to membrane damage and subsequent cellular toxicity. This research sought to determine if siRNA transfer could occur while the membrane barrier remains intact.

Significance & Outlook

These findings support a model where siRNA-lipid association facilitates the crossing of the membrane without sustained loss of barrier function. By establishing this cell-free platform, the researchers provide a method to dissect LNP-mediated RNA delivery and develop new formulations that improve delivery efficacy while limiting potential membrane damage.

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