Fig. 4: Magnitude of cytosolic delivery is determined by the siRNA-to-lipid ratio.
From: Single-cell quantification and dose-response of cytosolic siRNA delivery
HeLa cells stably expressing d1-eGFP were treated with lipoplexed AF647-siRNA targeting eGFP (siGFP-1 or siGFP-2) and analyzed by a flow cytometry or b–f live-cell microscopy followed by single-cell analysis. a d1-eGFP knockdown evaluated after 24 h siRNA-lipoplex incubation. Mean ± s.d. is shown. Relative and |absolute| IC50-values are indicated. N = 3 (siGFP-1) and 4 (siGFP-2) independent experiments. b Magnitudes of siRNA releases using lipoplexes formulated with different amounts siGFP-1 or siGFP-2 (constant for siLuc). The solid line is the median and the dashed lines are the 25th and 75th percentiles. N = cells indicated above violins. c Magnitude and time of siRNA release events during incubation with 200–666 pM lipoplexed AF647-siRNA. Lipoplexes were formed with a constant concentration and siRNA:lipid ratio (2:4 pmol:µl) with different volumes added to cells. The start of the experiment is t = 0. Single-cell measurements are shown, N = 151, 84, and 143 cells from three, one, and four independent experiments, respectively. d Cumulative frequency of siRNA release events in cells incubated with indicated concentrations of lipoplexed siGFP-1, siGFP-2 or siLuc. Mean values are shown. N = 2, 1, and 5 independent experiments, respectively. e Peak cytosolic siRNA concentrations estimated by release modeling and corresponding goodness-of-fit statistics (R2). Individual release events are shown. N = 546 and 316 events from 13 and 6 independent experiments, for siGFP-1 and siGFP-2, respectively. f d1-eGFP knockdown following one (solid lines) or multiple (dashed lines) siRNA release events per cell. Typically, after a second event is detected, subsequent measurements are excluded from analysis (solid lines). The first event occurs at t = 0. The line is the mean, shaded area is an 80% confidence interval. N = 546 and 316 cells from 13 and 6 independent experiments for siGFP-1 and siGFP-2, respectively. Source data are presented in the Source Data file.
