Fig. 3. Low pH activated endo/lysosomal escape.

a, Typical confocal images of MDA-MB-453 TNBC cells incubated with Cy5.5-siPol2@NPs (with CO₂) (nanoparticles encapsulated with Cy5.5 labelled siPol2 and CG-CO₂) and Cy5.5-siPol2@NPs (without CO₂) (nanoparticles encapsulated with Cy5.5 labelled siPol2 and CG) for 1, 3, and 6 h at 37 °C. The cell nuclei were stained using DAPI (blue), the endo/lysosomes were stained using LysoTracker Green (green), and siPol2 were labelled with Cy5.5 (red); DIC represents differential interference contrast. For the Cy5.5-siPol2@NPs (without CO₂) group, the green and red fluorescence overlaps at all the time points. For the Cy5.5-siPol2@NPs (with CO₂) treatment, the overlap is reduced at all the three time points and minimal at 6 h. Scale bars: 20 μm and 5 μm for low and high magnification images, respectively. The experiments were repeated three times independently. b, Quantitative analysis of co-localization of Cy5.5-siPol2 with endo/lysosomes labelled with LysoTracker Green. Manders’ Coefficient M1 denotes the fraction of Cy5.5-siPol2 overlapping with LysoTracker Green, and M2 denotes the fraction of LysoTracker Green overlapping with Cy5.5-siPol2. The coefficients are close “1” if they are highly co-localized (n= 10 images from three independent experiments). Error bars denote mean ± s.d., *: p < 0.05 and ***: p < 0.001. The statistical significance was assessed by Student t-test (paired, two tailed) and one-way ANOVA with a Dunnett’s host-hoc test for M1 and M2, respectively.