Poly(2‐ethyl‐2‐oxazoline) (POx) as Poly(ethylene glycol) (PEG)‐Lipid Substitute for Lipid Nanoparticle Formulations

Polyoxazolines have long been considered as promising alternatives to poly(ethylene glycol) (PEG) due to their comparable properties, in particular regarding their stealth effect toward the immune system. Lipid nanoparticles (LNPs), as utilized, e.g., in the COVID-19 vaccines, contain PEG-lipids. However, alternatives are required because of the "PEG dilemma" recognized by an increase in anti-PEG antibodies in the human population. In this study, poly(2-ethyl-2-oxazoline) (PEtOx)-based lipids with different degrees of polymerization are synthesized and subsequently used to formulate mRNA-loaded LNPs. The effect of polymer chain length on the size, immunoreaction, and transfection efficiency is investigated in detail. In addition, in-depth transfection studies are performed using super-resolution microscopy (SRM) to investigate the uptake mechanism of PEtOx-based LNPs in comparison to PEG-LNPs. These combined approaches are utilized to identify the best performing LNP, being superior to the commercial PEG-lipid used in the Comirnaty formulation.