Preparation of Antibody-Modified mRNA-LNPs via a Combination of Post-Encapsulation and Post-Insertion

Using ligand-modified lipid nanoparticles (LNPs) to encapsulate messenger RNA (mRNA) has become an important modality for tissue/organ/cell-specific gene delivery. Modifying mRNA-LNPs either with antibodies or their derivatives (Ab-mRNA-LNPs) has emerged as a promising drug delivery system (DDS) due to its efficacy and target specificity. Manufacturing Ab-mRNA-LNPs, however, involves complex and multi-step procedures that include the encapsulation of mRNA followed by the anchoring of antibodies via polyethylene glycol (PEG) linker-conjugated lipids. In this work, we present a seamless strategy for the production of Ab-mRNA-LNPs by integrating the post-encapsulation of mRNA into mRNA-free LNPs (fLNPs) with a post-insertion of ligand-conjugated PEG-lipid (Lig-PEG-lipid). Using a T-cell-targeting model, we further describe how targeting efficacy is influenced by controlling the surface nature of the Ab-mRNA-LNPs either via the modification of PEG-lipids or by replacing the phospholipids. Modification of the DSPE-PEG-methoxy and employment of either DSPC or DPPC significantly improves the accumulation of Ab-mRNA-LNPs in T cells. These findings highlight the importance of LNP surface engineering.