Stabilizing Anionic mRNA Lipid Nanoparticles by Cleavable Crosslinking of Cholesterol

Area of Science:

• Biotechnology
• Nanomedicine
• Molecular Biology

Background:

• Lipid nanoparticles (LNPs) are crucial for mRNA delivery, but their stability and anionic lipid composition limit formulation design.
• Weak RNA-lipid interactions can cause LNP rearrangement, reducing efficacy, especially for spleen-targeted delivery.

Purpose of the Study:

• To enhance the stability of anionic LNPs using a cleavable crosslinking strategy without altering core lipid components.
• To expand the accessible formulation space for tissue- and cell-specific mRNA delivery.

Main Methods:

• Developed and optimized a cleavable crosslinking strategy by tuning crosslinker and PEG-diamine lengths.
• Assessed structural stability and splenic mRNA expression of crosslinked versus uncrosslinked anionic LNPs.
• Performed mechanistic analyses to understand transfection efficiency improvements.

Main Results:

• Achieved balanced structural stability in anionic LNPs through tunable crosslinking.
• Demonstrated significantly increased splenic mRNA expression at 12 hours post-administration in crosslinked LNPs.
• Identified T cells as a major cell type transfected (33.2% of CD45+ tdTomato+ cells in spleen).

Conclusions:

• Tunable crosslinking effectively stabilizes anionic LNPs, improving mRNA delivery to the spleen.
• This strategy expands the design landscape for targeted mRNA delivery systems.
• Controlled mRNA release and intracellular processing are key to enhanced transfection efficiency.