PEG-Free Tunable Poly(2-Oxazoline) Lipids Modulate LNP Biodistribution and Expression In Vivo after Intramuscular Administration

Over the past decade, lipid nanoparticles (LNPs) have emerged as a transformative delivery platform, particularly in the field of mRNA vaccines, by enabling the stabilization and efficient intracellular delivery of nucleic acids. Importantly, the FDA approved LNPs used in the Pfizer-BioNTech and Moderna COVID-19 vaccines, rely on polyethylene glycol (PEG) to stabilize the nanoparticle in vivo . However, recent studies revealed that anti-PEG antibodies are ubiquitous in the population and are known to be a major cause of anaphylaxis and reduced therapeutic efficacy by accelerating blood clearance of PEGylated products. In this study, we report the development of novel poly(2-oxazoline) (POx) “stealth” lipids as PEG alternatives for LNP formulation. POx polymers are known to be poorly immunogenic and mitigate accelerated blood clearance. Upon varying polymer hydrophilicity and molecular weight, we screened transfection efficiency in multiple cell lines and evaluated top candidates in vivo by intramuscular administration. Our findings demonstrate that POx-lipids incorporating shorter hydrophilic methyl- and ethyl-oxazoline POx chains enhance transfection both in vitro and in vivo , while concurrently reducing liver accumulation and improving dendritic cell uptake - key features for effective vaccine delivery. These POx-lipids boost expression in muscle tissue by up to threefold, and greatly increase accumulation in extrahepatic tissues, including lymph nodes and spleen - critical sites for immune priming in the in the context of vaccination. Altogether, this work marks a notable advancement toward replacing PEG in LNP-based therapies and provides a clear path to optimizing POx-lipids for targeted vaccine and therapeutic applications.