Degradable PEG-lipids for lipid nanoparticle mRNA formulation

Lipid nanoparticles (LNPs) are non-viral carriers which are widely used for the delivery of nucleic acid based drugs, including mRNA, which are prone to degradation, sub-optimal pharmacokinetics and poor intracellular delivery in unformulated from. [1] Such LNPs are generally composed of an ionizable cationic lipid, cholesterol, a phospholipid, and a PEGylated lipid (PEG: poly(ethylene glycol)). The latter is crucial for limiting LNP size, colloidal stability and influencing biodistribution in vivo. PEG is a non-degradable polymer and has limited options to introduce functionality along its backbone. [2] Here we demonstrate a straightforward route which concomitantly introduces degradability and functionality onto existing PEG and PEG-lipids and demonstrate its usefulness for mRNA LNP formulation. The commercially available PEG lipid DMG-PEG (1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000) was modified in a single step with either a carboxylic acid-functionalized TAD (acidTAD) or a butyl-functionalized TAD (buTAD) compound. Under irradiation of 520 nm light, a hemi-aminal bond is formed between the TAD and the ether repeating unit in PEG [3]. Degradation of the hemi-aminal in aqueous medium leads to low molecular weight PEG oligomers with reaction kinetics depending on pH and temperature, showing accelerated kinetics at 40 °C at both neutral and acidic pH, whereas slower kinetics were observed at room temperature and basic pH. The obtained DMG-PEG-acidTAD and DMG-PEG-buTAD could successfully be used for mRNA LNP formulation, showing similar size and encapsulation efficiency compared to native DMG-PEG, albeit with a negative zeta-potential due to the chemical nature of the TAD-conjugate. In vitro evaluation on 2D cell cultures showed lack of toxicity of the TAD-modification and increased mRNA transfection efficiency of the LNP without increasing LNP uptake. Studies to elucidate the underlying reason for the improved transfection efficiency and the influence of TAD-modification on in vivo biodistribution and mRNA transfection are currently ongoing.