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 develop a cleavable crosslinking strategy to stabilize anionic LNPs without altering core lipid components.
• To enhance the structural integrity and in vivo performance of anionic LNP formulations for mRNA delivery.

Main Methods:

• A cleavable crosslinking strategy was applied to anionic LNPs using tunable cholesterol-derived crosslinkers and PEG-diamines.
• Structural stability was optimized by adjusting crosslinker and PEG-diamine lengths.
• Splenic mRNA expression and cellular uptake were evaluated in vivo.

Main Results:

• Optimized crosslinked LNPs demonstrated enhanced structural stability compared to uncrosslinked counterparts.
• A significant increase in splenic mRNA expression was observed at 12 hours post-administration.
• Mechanistic studies indicated controlled mRNA release and altered intracellular processing, leading to improved transfection efficiency, with T cells showing significant tdTomato expression.

Conclusions:

• A tunable crosslinking method effectively stabilizes anionic LNPs, overcoming previous limitations in formulation design.
• This approach expands the accessible design space for tissue- and cell-specific mRNA delivery applications.
• The findings pave the way for more robust and targeted mRNA-based therapeutics.