Polysarcosine-Functionalized mRNA Lipid Nanoparticles Tailored for Immunotherapy

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.

Keywords: LNPs; cancer; flow cytometry; immunotherapy; lipid nanoparticles; mRNA; polysarcosine; small-angle X-ray scattering; vaccine.

Figure 1

Small-angle X-ray scattering (SAXS) investigation. (A) SAXS patterns of different LNP formulations in phosphate buffer (pH 4.5, light blue) and application buffer (pH 5.7, dark blue). Formulations are displayed according to their composition with PEG-grafted LNPs (left) and pSar-grafted LNPs (right) and their ionizable and helper lipid are displayed right to the scattering patterns. Scattering patterns are vertically shifted for better visualization. (B) Comparison between LNP formulations in d-spacing (top). Investigated pairs generated with formulations only differing in one lipid component (ionizable lipid, helper lipid, stealth lipid). Mean of the differences in the compared formulations is shown on the right and represents the mean factor in which the formulations differ in d-spacing when comparing the investigated pairs; the same procedure for correlation length is at the bottom. Data displayed as mean ± S.D.

Figure 2

In vitro tolerability of LNP formulation 1–8 in human peripheral blood mononuclear cells (hPBMC). Dose ranged from 100 ng to 2000 ng. Viability of each LNP formulation is shown as %Viable hPBMC. Data are presented as mean ± S.D., n = 3 technical replicates per LNP formulation.

Figure 3

In vitro dose-dependent transfection efficiency of Monocytes for LNP formulations 1–8. Dose ranged from 100 ng to 2000 ng. Transfection efficiency of each LNP formulation is shown as %Thy1.1+ Monocytes. Data are presented as mean ± S.D., n = 3 technical replicates per LNP formulation.

Figure 4

In vitro transfection efficiency of Thy1.1 RNA containing LNPs at a dose of 1000 ng in hPBMC, Monocytes as representative cell group. (A) Thy1.1-expressing Monocytes analyzed by flow cytometry. Numbers indicate the percentage of Thy1.1+ Monocytes. (B) Transfection efficiency of all PEG-lipid versus pSar-lipid LNPs shown as %Thy1.1+ Monocytes. Data are presented as mean ± S.D., analyzed by a two-way ANOVA with Šidák’s multiple comparison test, **** p < 0.0001, n = 3 technical replicates per LNP formulation.

Figure 5

In vitro cell-binding studies of Cy5-labeled RNA containing DODMA-LNP at a dose of 1000 ng in hPBMCs. (A) Cy5-labeled RNA-positive Monocytes analyzed by flow cytometry. Numbers indicate the percentage of Cy5+ Monocytes. (B) Cell-binding efficiency of each DODMA-LNP formulation is shown as %Cy5+ Monocytes. Data are presented as mean ± S.D., analyzed by a two-way ANOVA with Šidák’s multiple comparison test, *** p < 0.001, **** p < 0.0001, n = 3 technical replicates per LNP formulation.