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. 2019 Sep 24;10:4333. doi: 10.1038/s41467-019-12275-6

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — Delivery of hEPO mRNA to cells via LNPs and analysis of endo-EVs. MC3-LNPs and DD-LNPs containing 100 μg of hEPO-mRNA were transferred to human epithelial (HTB-177) cells. Untreated cells and cells treated with empty LNPs (without hEPO mRNA) were used as controls. a Amount of hEPO mRNA detected in cells. Untreated (n = 9), DD-LNPs (w/o mRNA) (n = 3), MC3-LNPs (w/o mRNA) (n = 3), DD-LNPs + mRNA (n = 7), and MC3-LNPs + mRNA (n = 5). b Amount of hEPO protein detected in cells. Untreated (n = 3), DD-LNPs (w/o mRNA) (n = 6), MC3-LNPs (w/o mRNA) (n = 3), DD-LNPs + mRNA (n = 5), and MC3-LNPs + mRNA (n = 3). c Amount of hEPO protein detected in the supernatant of cultured cells. Untreated (n = 3), DD-LNPs (w/o mRNA) (n = 6), MC3-LNPs (w/o mRNA) (n = 3), DD-LNPs + mRNA (n = 5), and MC3-LNPs + mRNA (n = 3). d Percentage of hEPO mRNA detected in the cytosol of cells relative to the total amount of mRNA administered (100 µg) to cells via LNPs. Untreated (n = 10), DD-LNPs (w/o mRNA) (n = 6), MC3-LNPs (w/o mRNA) (n = 3), DD-LNPs + mRNA (n = 8), and MC3-LNPs + mRNA (n = 7). e Hypothetical presentation of the endosomal escape of hEPO mRNA of LNPs into the cytoplasm and translation into protein, versus loading of hEPO mRNA into endo-EVs. f Total amount of hEPO mRNA quantified in endo-EVs isolated from LNP-treated cells. Untreated (n = 3), DD-LNPs (w/o mRNA) (n = 3), MC3-LNPs (w/o mRNA) (n = 3), DD-LNPs + mRNA (n = 10), and MC3-LNPs + mRNA (n = 5). g Molar concentrations of ionizable lipids and hEPO-mRNA (ionizable lipids per hEPO mRNA nucleotides) in originally formulated LNPs (control, n = 1), which contains 3 moles of ionizable lipids per 1 mole of mRNA nucleotides. h Molar concentration of ionizable lipids and hEPO-mRNA of mc3-EVs (n = 7). i Molar concentration of ionizable lipids and hEPO-mRNA of dd-EVs (n = 6). j Stoichiometric comparison between LNPs and endo-EVs regarding molar ratio (mole/mole) of ionizable lipids per hEPO-mRNA nucleotides. Red circles (dd-EV) and blue circles (mc3-EV). Ionizable lipids and hEPO-mRNA of mc3-EVs (n = 7) each. Ionizable lipids and hEPO-mRNA of dd-EVs (n = 6) each. Data are presented as scatter dot plots including the mean (bars) and standard deviation (SD) of the number (n) of biologically independent samples specified for each panel. MC3-LNP and DD-LNP groups (a–d, f) were compared using the unpaired two-tailed Student’s t-test. **p < 0.01, ****p < 0.0001 and ns = not significant. Source data are provided as a Source Data file