A robust post-insertion method for the preparation of targeted siRNA LNPs.

Area of Science:

• Biotechnology
• Nanomedicine
• Molecular Biology

Background:

• Targeted nucleic acid delivery enhances therapeutic efficacy and allows treatment of previously undruggable targets.
• Current methods for targeted delivery vehicles often use liposomes, which differ structurally from lipid nanoparticles (LNPs).
• Existing preparation protocols for liposomes may not directly translate to siRNA LNP preparation.

Purpose of the Study:

• To develop a robust and facile method for preparing targeted or fluorescently labeled siRNA LNPs.
• To evaluate post-insertion of ligand-lipid conjugates as a superior method compared to direct surface modification.
• To optimize the preparation process for controlled decoration of siRNA LNPs with ligand combinations.

Main Methods:

• Utilized copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) for post-insertion of ligand-lipid conjugates into preformed LNPs.
• Investigated the critical role of solvent removal timing during dialysis on LNP hydrodynamic diameter and polydispersity index (PDI).
• Generated siRNA LNPs with combined targeting and fluorescent tracking ligands.

Main Results:

• Post-insertion of ligand-lipid conjugates into preformed LNPs preserves physicochemical parameters better than direct surface modification.
• Post-insertion after dialysis minimizes increases in hydrodynamic diameter and PDI.
• Ligand-lipid conjugate post-insertion demonstrated rapid kinetics and high efficacy across a wide temperature range.
• Successfully generated siRNA LNPs with both targeting and fluorescent ligands to monitor uptake kinetics.

Conclusions:

• Described a robust and facile approach for generating targeted and labeled siRNA LNPs.
• Post-insertion is a superior strategy for decorating LNPs with targeting ligands.
• Optimized protocol enables controlled and facile decoration of siRNA LNPs with diverse ligand combinations for advanced nucleic acid therapies.