3.3. Mechanism study of complex uptake
The intracellular delivery mechanism of the S-R15 complex was analyzed using endocytosis inhibitors and flow cytometry (Fig. 6). Graphs showed cell distributions without an inhibitor of the red line and with an inhibitor of the green line, respectively. The images of the first row represented the penetrating inhibition of FITC-labeled S-R15 (Fig. 6). When the reference point was taken at 89.46% in red distribution, the cell population decreased to 53.14% in clathrin-meditated endocytosis inhibitor chlorpromazine and 68.33% in lipid raft-mediated endocytosis inhibitor methyl-β-cyclodextrin. In contrast, the cell populations showed no decrease in the cases of phagocytosis/micropinocytosis inhibitor cytochalasin D and caveolae-meditated endocytosis inhibitor Filipin III. Thus, the penetration of FITC-labeled S-R15 was inhibited by clathrin-mediated endocytosis dominantly and lipid raft-mediated endocytosis secondly.
The images of the second row represented the penetrating inhibition of Cy3-labeled siRNA. When the reference point was set at 89.26% in red distribution, the cell population decreased to 68.20% prominently in lipid raft-mediated endocytosis inhibitor methyl-β-cyclodextrin. In contrast, the other cell populations showed no decrease, except for methyl-β-cyclodextrin. Therefore, the permeation of Cy3-labeled siRNA was inhibited by lipid raft-mediated endocytosis dominantly. In summary, both experiments using FITC-labeled S-R15 and Cy3-labeled siRNA showed consistency of inhibition by lipid raft-mediated endocytosis inhibitor methyl-β-cyclodextrin. Besides, penetration of FITC-labeled S-R15 was inhibited by the clathrin-mediated endocytosis.