Formulation methods for peptide-modified lipid nanoparticles

Lipid nanoparticles (LNPs) are a promising non-viral gene carrier, but one significant unmet challenge is cell-specific delivery in extrahepatic organs. Peptides are one class of targeting ligand that have been used to target nanoparticles, including LNPs. Herein, we compared two formulation approaches that use the polyethylene glycol (PEG)-lipid to display targeting peptides: (1) post-conjugation targeted (PCT), in which LNPs were formulated with PEG-lipid with chemical handles and subsequently modified with peptides, and (2) in-line targeted (ILT), in which peptide-PEG-lipid conjugates were directly used in LNP formulation. We observed that PCT and ILT LNPs had similar physicochemical properties, but ILT LNPs aggregated when formulated with a large peptide. Using cyclic RGD as a model ligand, we observed that while binding and uptake of LNPs in cultured cells were similar between approaches, PCT LNPs led to higher activity. Systemic administration revealed that LNPs formulated with both methods led to shifts in organ biodistribution compared to untargeted LNPs, but PCT resulted in higher transfection compared to ILT. Finally, analysis of cell tropism showed that the transfection activity of cRGD LNPs was shifted towards endothelial cells in multiple organs. We conclude that while PCT LNPs required more processing steps over the ILT LNPs, they led to superior formulations that led to active peptide targeting.