Fig. 4

Analysis of PLGA-PEI PCS NPs internalization in MSCs. a Pitstop2 and Dynasore were used to analyze the mechanism of internalization. b FACS analysis revealed approximately 40% inhibition by Dynasore. N.C.: No treatment, P.C.: 5 μg/mL PLGA-PEI PCS NPs. c Immunofluorescence imaging analysis of the endocytic pathway of PCS NPs. The early endosome marker EEA1, the late endosome marker Rab7, and the Golgi apparatus marker GM130 were observed for 15 min, 30 min, 60 min, and 6 h after the MSCs were exposed to PCS NPs. Red florescence indicates PCS NPs and green florescence depicts EEA1 (first row), Rab7 (second row), and GM130 (third row). White arrows indicate the colocalization between NPs and cellular organelles. d Fluorescent images were taken after 30 min, 60 min, and 24 h to observe the colocalization between NPs and lysosomes. Red florescence indicates PCS NPs and green florescence represents LysoTracker Green detected in lysosomes. White arrows depict the merged areas between NPs and cellular organelles. e Characterization of endocytic pathway of PCS NPs using Rab7 inhibitor. No NPs are internalized, even after 6 h, in the presence of the inhibitor. PCS NPs are labelled red, while Rab7 is stained green. f PLGA-PEI PCS NPs are not detected inside the cells after 30 min, 1 h, and 6 h incubation periods with Rab7 inhibitor. Data are mean ± SD. *p < 0.05, **p < 0.05, ***p < 0.005