Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy

Pharmaceutics, vol. 15, pp. 2068

Abstract

Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions.

Authors 11

1. Christoph Wilhelmy

   Johannes Gutenberg University Mainz

   Affiliation as printed

   Department of Biopharmaceutics and Pharmaceutical Technology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany

2. Isabell Sofia Keil

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz · Translationale Onkologie an der Universitätsmedizin der Johannes Gutenberg-Universität Mainz

   Affiliation as printed

   TRON—Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, 55131 Mainz, Germany

3. Lukas Uebbing

   Johannes Gutenberg University Mainz

   Affiliation as printed

   Department of Biopharmaceutics and Pharmaceutical Technology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany

4. Martin A. Schroer

   Deutsches Elektronen-Synchrotron DESY · European Molecular Biology Laboratory · European Molecular Biology Laboratory · University of Duisburg-Essen

   Affiliation as printed

   European Molecular Biology Laboratory (EMBL) Hamburg Outstation, c/o DESY, 22607 Hamburg, Germany

   Nanoparticle Process Technology (NPPT), Faculty of Engineering, University of Duisburg-Essen, 47057 Duisburg, Germany

   European Molecular Biology Laboratory (EMBL) Hamburg Outstation, c/o DESY, 22607 Hamburg, Germany; Nanoparticle Process Technology (NPPT), Faculty of Engineering, University of Duisburg-Essen, 47057 Duisburg, Germany

5. Daniel Franke

   Deutsches Elektronen-Synchrotron DESY · European Molecular Biology Laboratory · European Molecular Biology Laboratory

   Affiliation as printed

   BIOSAXS GmbH, 22607 Hamburg, Germany

   European Molecular Biology Laboratory (EMBL) Hamburg Outstation, c/o DESY, 22607 Hamburg, Germany

   BIOSAXS GmbH, 22607 Hamburg, Germany; European Molecular Biology Laboratory (EMBL) Hamburg Outstation, c/o DESY, 22607 Hamburg, Germany

6. Thomas Nawroth

   Johannes Gutenberg University Mainz

   Affiliation as printed

   Department of Biopharmaceutics and Pharmaceutical Technology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany

7. Matthias Barz Aachen LACDR—Leiden Academic Centre for Drug Research

   Leiden University · Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Department of Dermatology, University Medical Center, Johannes Gutenberg University Mainz, 55131 Mainz, Germany

   LACDR—Leiden Academic Centre for Drug Research, Leiden University, 2333 Leiden, The Netherlands

8. Uğur Şahin

   Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz

   Affiliation as printed

   Department of Immunology, University Medical Center, Johannes Gutenberg University Mainz, 55131 Mainz, Germany

9. Heinrich Haas

   Johannes Gutenberg University Mainz · BioNTech (Germany)

   Affiliation as printed

   BioNTech SE, 55131 Mainz, Germany

   Department of Biopharmaceutics and Pharmaceutical Technology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany

   BioNTech SE, 55131 Mainz, Germany; Department of Biopharmaceutics and Pharmaceutical Technology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany

10. Mustafa Diken corresponding

    Johannes Gutenberg University Mainz · University Medical Center of the Johannes Gutenberg University Mainz · Translationale Onkologie an der Universitätsmedizin der Johannes Gutenberg-Universität Mainz

    Affiliation as printed

    TRON—Translational Oncology at the University Medical Center of Johannes Gutenberg University gGmbH, 55131 Mainz, Germany

11. Peter Langguth corresponding

    Johannes Gutenberg University Mainz

    Affiliation as printed

    Department of Biopharmaceutics and Pharmaceutical Technology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany