Fig. 3: LNPs damage Rab5⁺ EEA1^+/– early endosomes to promote cytosolic RNA release.

HeLa-Gal9-YFP cells expressing mScarlet- or mCherry-tagged markers of endosomal compartments were incubated with 50 nM AF647-siRNA-LNP or 0.75 μg mL^–1 Cy5-mRNA-LNP. Images were acquired with a high-speed widefield microscope, and events with de novo formation of galectin-9 foci were identified. a Galectin-9 is recruited to a siRNA-LNP containing endosome marked by EEA1, and b a mRNA-LNP containing endosome marked by Rab5. Images are representative of 109 EEA1⁺ and 74 Rab5⁺ events, from 2 and 4 independent experiments, for siRNA- and mRNA-LNP respectively. Scale bar is 2 µm. c LNP⁺ endosomes showing galectin-9 recruitment were tracked in 4D, measuring the endosomal marker fluorescence intensity. A dynamic intensity threshold (0.5 × 90th percentile intensity per cell) was applied to determine the fraction of vesicles with presence of the respective markers at a ~ 10 s time-interval centered around start of galectin-9 recruitment. Bars show mean ± s.d. of 2–5 independent experiments (circles) per condition. Number of events (N_E) and experiments (N_X) shown in figure in parentheses as (N_E;N_X). Number of analyzed cells (order as c): 27, 23, 52, 14, 29, 15 and 25, 27, 13 cells for siRNA- and mRNA-LNP respectively and the indicated endosomal marker. d Rab5 or EEA1 fluorescence intensity measured on damaged LNP⁺ and Rab5⁺ or EEA1⁺ vesicles. Values were normalized to the 90th percentile marker intensity per cell (representing a typical marker-labeled object), and aligned in time so that t = 0 is the first time-point with galectin-9 recruitment. N = vesicles, as indicated. Lines are mean; shades are 95% CI of mean. e Heatmaps showing all individual vesicles analyzed in (c) and (d). Endosomes are grouped as compartment marker^– or marker⁺ (left and right vertical subdivisions of maps, respectively), as described in (c). All traces were aligned in time (vertical) as described in (d). N = vesicles as shown in (c).