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. 2022 Feb 20;9(12):2200173. doi: 10.1002/advs.202200173

Figure 4.

Figure 4

In vitro transcellular transport of micelles. a) The exocytosis kinetics measured by flow cytometry. ECDHCC‐1 cells were incubated with Cy5‐labeled micelles for 3 h and then re‐cultured in a fresh medium for timed intervals before flow cytometric analysis. b) Transepithelial transport of Cy5‐labeled micelles across the ECDHCC‐1 cell monolayer on a transwell membrane. c) Cumulative transport of the micelles. The Cy5‐labeled micelles at a Cy5‐eq. dose of 0.5 µg mL−1 were added onto the apical side, and the micelle concentrations in the basolateral compartment were measured at timed intervals by detecting the Cy5 fluorescence. d) Cellular uptake of the transported micelles in MCF‐7/ADR cells on the basolateral side after 24 h incubation. Cy5‐eq. dose: 0.5 µg mL−1. Scale bars: 100 µm. Cell nuclei were shown in blue and Cy5 in red. e) Cy5 fluorescence intensity of MCF‐7/ADR cells quantified by flow cytometry. f) Intercellular transport of Cy5‐labeled micelles between MCF‐7/ADR cells. The cells (first batch) were cultured with micelles at a Cy5‐eq. dose of 1 µg mL−1 for 6 h, washed with PBS, and imaged (i); the cells were then cultured in fresh medium for 12 h, and the medium was harvested to incubate the second batch of cells for 12 h, followed by washing and imaging (ii); the same procedures were implemented for another two rounds (iii and iv). Scale bar: 50 µm. g) Distribution of PEG‐Cy5PCL, OPDMA‐Cy5PCL, and OPDEA‐Cy5PCL in MCF‐7/ADR MTSs and the effects of endo/exocytosis inhibitors on the distribution. The MTSs were pretreated with or without cytochalasin D (5 × 10−6 m) or brefeldin A (90 × 10−6 m) for 10 h and then exposed to different DOX formulations for 4 h (Cy5 eq. dose of 5 µg mL−1). The MTSs were washed twice with 10% heparin‐containing PBS and visualized using confocal microscopy in Z‐stacks with 30 µm intervals. The midplane is denoted as 0 µm. Scale bar: 250 µm. h) The Cy5 fluorescence intensity along with the randomly selected white arrows across OPDMA‐Cy5PCL and OPDEA‐Cy5PCL‐treated MTSs. * p < 0.05, ** p < 0.01.