Fig 1: Uptake and lysosomal co-localization of protein-LNP complexes in HepG2 cells.a HepG2 cells internalizing LNPs loaded with Cy5-mRNA pre-incubated with high-binding corona proteins were visualized by confocal microscopy. Representative image of LNP + VTN incubations showing LNPs (Cy5; red), cell membrane (CellBrite membrane dye; green), and nuclei (Hoechst; blue). b Inset showing a magnified view of the region outlined by the red box in (a). c Quantification of Cy5 (LNP) signal per cell demonstrates differences in cell Cy5-mRNA uptake between select corona protein incubations (n = 4 technical replicates, n = 3 biological replicates). Each dot represents an individual field-of-view-level measurement, color-coded by biological replicate; larger, black-outlined dots indicate the mean value for each biological replicate. Statistical analysis was performed by a nested one-way ANOVA test followed by Dunnett’s multiple comparisons test. d, e Cy5 signal from HepG2 cells internalizing LNPs loaded with Cy5-mRNA pre-incubated with high-binding corona proteins was also quantified by flow cytometry. d Percentage of Cy5-positive cells and e mean fluorescence intensity show uptake trends consistent with microscopy. Data points shown are biological replicates (n = 3 technical replicates, n = 3 biological replicates). Error bars denote standard deviation. Statistical analysis was performed by repeated measures one-way ANOVA test with Geisser-Greenhouse correction, followed by Dunnett’s multiple comparisons test. To compare endosome entrapment for select protein incubations, co-localization of the Cy5 signal (LNP) and fluorescently labeled lysosomes (green) was analyzed. Representative image of LNP + ApoE incubation shows f LNPs (red), lysosomes (green) and nuclei (blue) fluorescently labeled. g Quantification of overlapping Cy5 (LNP) and lysosome signal per cell (n = 4 technical replicates, n = 4 biological replicates). Each dot represents an individual field-of-view-level measurement, color-coded by biological replicate; larger, black-outlined dots indicate the mean value for each biological replicate. Statistical analysis was performed by a nested one-way ANOVA test followed by Dunnett’s multiple comparisons test. For all statistical analyses performed, *, **, and *** represent p ≤ 0.05, 0.01, and 0.001, respectively. All image thresholding was applied uniformly across samples, and image channels were adjusted solely for visualization purposes.
Fig 2: In vitro mRNA expression with delivery by protein-LNP complexes.a LNPs loaded with mRNA encoding luciferase were incubated with selected high-binding corona proteins (0.05 ng mRNA: 1 ng protein) prior to introduction to HepG2 cells seeded at 4.7 × 104 cells per cm2 (100 ng mRNA per well). The luminescence was measured as a proxy for mRNA expression to understand the effect of proteins on LNP delivery efficiency. Luminescence was normalized to the average of each no-corona LNP biological control for all in vitro studies. Created in BioRender. Voke, E. (2025) https://BioRender.com/58u4s4t. b Resulting luminescence of pre-incubations of individual proteins with LNPs showed no significant change in luminescence (mRNA expression) for ApoE, A2M, or a mixture of the proteins, while showing a significant decrease for VTN or CR, each relative to the no-corona LNP control. c Dose–response of protein concentrations for VTN incubated LNPs showed a significant decrease in mRNA expression compared to the no-corona LNP control. d Cell viability showed no statistical difference for protein incubations. N = 4 technical replicates, n = 3 biological replicates. Data points shown are biological replicates. All data are presented as mean values with standard deviation. Statistical analysis was performed by repeated measures one-way ANOVA test with Geisser–Greenhouse correction, followed by Dunnett’s multiple comparisons test where * and ** represent p ≤ 0.05 and p ≤ 0.01, respectively.
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