Comparative Analysis of Poly(Ethylene Glycol) (PEG) and Poly(2‐Ethyl‐2‐Oxazoline) (PEtOx) for Organic Solvent‐Free Lipid Nanoparticle Formulations
ABSTRACT 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.
Authors
- Lucas Gath (ORCID: https://orcid.org/0009-0006-5112-7273)
- Ekaterina Tsarenko
- Christian Franke (ORCID: https://orcid.org/0000-0002-0326-7232)
- Aleksandar Rusevski (ORCID: https://orcid.org/0009-0008-0642-0483)
- Anja Traeger (ORCID: https://orcid.org/0000-0001-7734-2293)
- Oliver Werz (ORCID: https://orcid.org/0000-0002-5064-4379)
- Julian Kimmig (ORCID: https://orcid.org/0000-0002-4478-3874)
- Vivien Bachmann (ORCID: https://orcid.org/0009-0000-2246-1682)
- Matthias Bros (ORCID: https://orcid.org/0000-0002-4662-0542)
- Caroline T. Holick (ORCID: https://orcid.org/0009-0000-1724-2725)
- Michael W Bauer (ORCID: https://orcid.org/0000-0002-1521-3514)
- Stephanie M. Schubert (ORCID: https://orcid.org/0000-0001-9527-7763)
- Ulrich S. Schubert (ORCID: https://orcid.org/0000-0003-4978-4670)
- Adrian Tibor Press (ORCID: https://orcid.org/0000-0002-6089-6764)
- Ivo Nischang (ORCID: https://orcid.org/0000-0001-6182-5215)
- Stephan Culemann (ORCID: https://orcid.org/0000-0001-9837-2811)
- Stephanie Hoeppener (ORCID: https://orcid.org/0000-0002-5770-5197)
- Claudia Waskow (ORCID: https://orcid.org/0000-0003-3261-0922)
- Laura J. Rosenberger (ORCID: https://orcid.org/0009-0004-8477-7892)
- Michael Streiber (ORCID: https://orcid.org/0009-0005-1385-6478)
- Stavroula Litsiou (ORCID: https://orcid.org/0009-0001-6599-2173)
- Lukas Harder
Institutions
- Helmholtz Institute Jena (DE)
- Helmholtz-Zentrum Berlin für Materialien und Energie (DE)
- University Medical Center of the Johannes Gutenberg University Mainz (DE)
- Leibniz Institute on Aging - Fritz Lipmann Institute (FLI) (DE)
- Jena University Hospital (DE)
- University Hospital in Halle (DE)
- Center for Sepsis Control and Care (DE)
- Martin Luther University Halle-Wittenberg (DE)
- Friedrich Schiller University Jena (DE)
Publication Details
- Journal
- Angewandte Chemie International Edition
- Published
- 2026-10-27
- DOI
- https://doi.org/10.1002/anie.5724119
- Primary Topic
- RNA Interference and Gene Delivery
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Carl-Zeiss-Stiftung
- Joachim Herz Stiftung
- Australian Government
- Freistaat Thüringen
- Deutsche Forschungsgemeinschaft
- Thüringer Aufbaubank
- Thüringer Ministerium für Bildung, Wissenschaft und Kultur
- Bundesministerium für Wirtschaft und Energie
- Leibniz-Gemeinschaft