Fig. 3: In vitro successful siRNA delivery and target gene silencing by IPMs.

a Confocal microscopy images of HSCs co-incubated with IPM/Cy5-siNC formed by different IOs for 2 h, with DAPI labeling nucleus. b Flow cytometry assay demonstrating the highest cellular uptake efficiency of IPM (IOA2, PEG5000-PLA8000) (n = 3 cell culture wells). c The confocal images of the lysosomal escape experiments revealing a decline in the co-localization signals from 4 to 6 h, with LysoTracker Green labeling of lysosomes and Hochest 33342 labeling of nuclei. d Statistical analysis of Pearson’s R for co-localization of Cy5 signal with lysosomal signal. At 4 h, the Pearson’s R in the IPM (IOA2) group was <0.5, changing from moderate to low correlation (n = 3 cell culture wells). e Statistical analysis of Overlap R for co-localization of Cy5 signal with lysosomal signal (n = 3 cell culture wells). f TNS assay for the apparent pKa of the three blank carriers (n = 3 independent experiments). g In vitro RNAi using IPM encapsulating siHSP47 and siHMGB1 formed by PEG5000-PLA8000 and IOA2, IOLA4, IOLA8, with LNP serving as a positive control and LNP/siNC serving as a negative control (n = 3 cell culture wells). The qPCR results showing the effective silencing of the HSP47 (h) and HMGB1 (i) genes in vitro, with LNP and Lipo2000 serving as positive controls and LNP/siNC and no siRNA serving as negative controls (n = 3 cell culture wells). Data were analyzed by One-way ANOVA followed by Tukey test. Values represent mean ± standard deviation (SD).