Exploring the Effects of Incorporating Different Bioactive Phospholipids into Messenger Ribonucleic Acid Lipid Nanoparticle (mRNA LNP) Formulations

Figures

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  Figure 1. Schematic of bioactive phospholipid LNP library construction. (a) The LNP formulation library was constructed by adding 1 of the 7 bioactive phospholipids into the Base formulation at molar concentrations of 5%, 15%, and 30%. (b) Chemical structures of the bioactive phospholipid used in this study.

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  Figure 2. Physicochemical property assessment of all of the formulations in the library. The Base formulation was prepared as the control (0% bioactive phospholipid). (a) Heatmap illustrating the particle size of the formulations measured by DLS analysis. (b) Heatmap illustrating the polydisperse index of the formulations measured by DLS analysis. (c) Heatmap illustrating the zeta potential measured by ELS analysis. (d) Heatmap illustrating the EE measured by RiboGreen assay. Note that only formulations that demonstrated EE > 80% were included for the biological function test next. (e) Heatmap showing the predictor screening analysis result from JMP that determines how incorporating different kinds of bioactive lipids at different molar concentrations impacts the physicochemical properties of the particles including size, PDI, zeta potential and EE. Statistical analysis was conducted using the 2-way ANOVA test followed by Tukey’s multiple comparison test. Heatmap squares represent means for n = 3 replicates. Level of significance compared to the Base formulation (0%): *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

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  Figure 3. Hela cell transfection of all 14 formulations (5 μg of mRNA LNP eGFP) that showed >80% EE. The Base formulation was included as a control. (a) Transfection efficiency as determined by the viable eGFP+ cell frequency from flow cytometry. Statistical analysis was conducted using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Bars represent means ± SD, n = 3. (b) Mean fluorescence intensity of cellular eGFP expression normalized against the eGFP expression level from the Base formulation transfection. Up to an ∼2-fold increase could be observed. Statistical analysis was conducted using the ordinary one-way ANOVA test followed by Dunnett’s multiple comparison test. Bars represent means ± SD for n = 3. Level of significance compared to the Base formulation: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

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  Figure 4. Heatmap of cytokine response from human PBMC transfection of all 14 formulations (0.5 μg of mRNA LNP eGFP) using Meso Scale Discovery human U-plex kits. Fifteen cytokines were measured. The Base formulation was included as a control. All of the values are normalized against the untransfected condition (UT). (a) Heatmap of cytokine response from all the formulations. (b–e) Heatmap of cytokine response split by formulations that share a similar headgroup such that other structural variations and different molar concentrations could be better compared. (b) Heatmap of cytokine response from all the CL formulations as compared to the Base. (c) Heatmap of cytokine response from all the PA formulations as compared to the Base. (d) Heatmap of cytokine response from all the lyso PC formulations as compared to the Base. (e) Heatmap of cytokine response from all the PS formulations as compared to the Base. Statistical analysis was conducted when possible using the 2-way ANOVA test followed by Tukey’s multiple comparison test. Heatmap squares represent means for n = 3 for all of the cytokines except IL-6 and IFN-γ when only singlet samples after dilution were within the detection range.

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  Figure 5. Immunophenotyping of LNP+ hPBMCs of all 14 formulations (0.5 μg of SYTO 13 stained mRNA LNP eGFP). (a) LNP+ hPBMCs composition breakdown after the Base formulation transfection. (b) LNP+ hPBMCs CD8+ T cell proportion change under transfection using different formulations, with 15% PA 18:1 as an example. (c) LNP+ hPBMCs CD19+ B cell proportion change under transfection using different formulations, with 15% PS 18:1 as an example. (d) LNP+ hPBMCs monocyte-derived dendritic cell (Mo-DCs, CD11B+ CD14+ CD11C+ HLA-DR+) proportion change under transfection using different formulations, with 15% lyso PA 18:1 as an example. (b–d) Statistical analysis was conducted using the ordinary one-way ANOVA test followed by Tukey’s multiple comparison test. All of the bars represent means ± SD, n = 3. Level of significance: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.