Efficient, biocompatible, and flexible application of CRISPR-Cas9 remains a major challenge in gene therapy. This study addresses a novel formulation method, focusing on organic solvent-free lipid nanoparticles for the efficient delivery of CRISPR-Cas9 components. Therefore, novel stealth lipids comprising the lipid anchor cholesterol hemisuccinate conjugated to poly(2-ethyl-2-oxazoline) (PEtOx) were synthesized and evaluated as a promising in vitro alternative to commercially available poly(ethylene glycol) (PEG)-based lipids. Our study identified PEtOx-lipids as markedly more biocompatible compared to PEG-based analogues (12-fold reduction in cytotoxicity; 99% reduced hemolytic effect) with high efficiency for mRNA gene delivery. We demonstrate successful CRISPR-Cas9 activity ex vivo and efficient uptake of PEtOx nanoparticles by macrophages in vivo (66.5% pos. F4/80+ macrophages). Our data demonstrate that PEtOx-based stealth lipids are a promising alternative to PEG. The resulting LNPs, optimized for fully aqueous manufacturing, address key limitations of current LNP systems, including immunogenicity and the reliance on ethanol-based production processes.
