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. Author manuscript; available in PMC: 2024 Jun 9.
Published in final edited form as: Nat Biomed Eng. 2023 Dec 11;8(5):544–560. doi: 10.1038/s41551-023-01131-0

Fig. 6 |. Local transfection, cellular uptake and endosomal escape of mRNA LNPs.

Fig. 6 |

a, Schematic of different immune responses induced by mRNA LNPs. Transfected APCs translate, process and present antigen epitopes on MHC-I molecules to CD8+ T cells, while transfected non-APCs such as myocytes translate and release antigen for APCs to internalize and present antigen epitopes on MHC-II molecules to helper T cells. Some exogenous antigens are uptaken and presented on MHC-I molecules by the cross-presentation pathway. b,c, Makeup of transfected cells at the injection sites at 24 h post injection with GFP mRNA (mGFP)-loaded C10, D6 and F5 formulations. Flow cytometry was used to determine the ratios of non-immune and immune cells (b) and the relative abundance of each cell type (c). Immune cells, CD45+; epithelial cells, CD326+; endothelial cells, CD31+; muscle cells, desmin+; adipocytes, CD45−CD31−CD36+. d–h, In vitro evaluation of transfection or uptake efficiency by formulations C10, D6 and F5 in C2C12 cells and BMDCs. Flow cytometry was used to determine the ratios of mCherry mRNA-transfected C2C12 cells to transfected BMDCs in in vitro co-culture (d), transfection efficiency of LNPs containing fLuc mRNA in pure C2C12 cell culture (e), transfection efficiency of LNPs containing mCherry mRNA in pure BMDCs (f), transfection efficiency of LNPs containing mGFP in pure C2C12 cells (g) and uptake efficiency of LNPs containing Cy5-labelled mRNA in C2C12 cells (h). i,j, Quantitative Cellomics HCA of endosomal escape by different LNPs using C2C12-Gal8-GFP cells. Total number of Gal8 spots per well (i) or average number of Gal8 spots per cell as an indication of endosomal escape level (j). k, LysoTracker was used to identify the co-localization of fluorescent labelled lysosomes (Lysotracker) and LNPs containing Cy5-labelled mRNA in C2C12 cells in vitro. Data represent the mean ± s.e.m. from a representative experiment (n = 4 biologically independent samples for b–j) of two independent experiments. For boxplots, the box extends from the 25th to the 75th percentiles and the line in the middle of the box is plotted at the median. *P < 0.05, ***P < 0.001, ****P < 0.0001.