Fig. 3: The dual-targeting ability, tissue penetration, cellular uptake and retention capability of EiNS.

a, b Confocal images and (c) flow cytometric analysis illustrating the in vitro internalization of s-Cy3EiNS after 12 h of incubation with Huh7 and Hepa1-6 cells, with siYTHDF1 labeled with Cy3 (n = 3 independent experiments). Scale bar, 20 µm. d YTHDF1^Cy3+ ratio in Huh7 and Hepa1-6 cells treated with various formulations (n = 3 independent experiments). e Mean fluorescence intensity (MFI) ratio of YTHDF1^Cy3+ in Huh7 and Hepa1-6 cells across different treatment groups (n = 3 independent experiments). Confocal images showing subcellular localization in (f) Huh7 and (g) Hepa1-6 cells incubated with s-Cy3EiNS for 1, 3, and 6 h at 37 °C (n = 3 independent experiments). Nuclei were stained with DAPI (blue), endo/lysosomes with LysoTracker Green (green), and siYTHDF1 with Cy3 (red). h, i Quantitative co-localization analysis of S-Cy3 with endo/lysosomes labeled by LysoTracker Green (n = 3 independent experiments). j Schematic illustration of deep tumor penetration of s-Cy3EiNS in 3D tumorspheres. Created in BioRender. Y.Q. (2025) https://BioRender.com/r8jogdb. k Representative CLSM images showing enhanced penetration of sEiNS compared to Lipo/s in Huh7 and Hepa1-6 tumorspheres (n = 3 independent experiments). Scale bar, 100 µm. l The IVIS imaging and (m) fluorescence intensity quantification at various time points in mice with subcutaneous Hepa1-6 tumors (tumors marked with white circles, n = 3 mice per group). EiNS(-M2Φpep), EiNS without M2Φpep modification. Data are expressed as mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test (d, e, h, i), or two-way ANOVA (m). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.