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.