Fig. 4: Intracellular delivery and endosomal escape of nanoparticles.

a HeLa cells were exposed to β-sitosterol containing eLNPs at different doses (1–400 ng) and compared to cholesterol-containing LNPs for cellular uptake. Cy5-EGFP mRNA was used as a tracer, and high-content imaging was performed and quantified using the Cellomics software. (n = 3; mean ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. Significance was determined using nonlinear regression with ANOVA.) Source data are provided as a Source Data file. b Uptake kinetics of LNPs and eLNPs at 10 ng mRNA per well (0–24 h) in HeLa cells. c 1st derivative of uptake of LNP and eLNP demonstrates substantially higher rate of uptake and retention in HeLa over the 24 h post-transfection. d Endosomal escape was visualized using smFISH. Representative fluorescent images showing mRNA, LNP, and image analysis after delivery with LNP or eLNP in HeLa cells. e Quantitative image analysis of the number of cytosolic mRNAs (turquoise bars) compared to the number of LNPs per cell (magenta bars) after cellular delivery. f Ratio of cytosolic mRNA delivery to total LNP uptake indicative of endosomal escape. g–j 3D-DyPLoT was performed for high spatiotemporal resolution of nanoparticle transport. g Representative trajectories of internalized LNP and eLNP. The measured 3D position is plotted at 1 ms temporal resolution. h Histograms of the log-linear efficiency for LNP (blue) and eLNP (red), both of which display a bimodal distribution of stationary and mobile. i Population of the stationary and mobile fractions for LNP and eLNP. Population was measured by fitting the histograms in h to a two-term Gaussian and extracting the area under the two modes of the distribution. n = 156 (LNP) and 286 (eLNP); mean ± SD. p = 0.0009. Significance was determined using t-test. j Best fit distributions of LNP and eLNP, showing a clear increase in the mobile fraction for eLNP.