3.2 Cellular uptake and internalization of NCs
Cellular uptake efficiency via R11-, RS- and RT-NCs was
checked using the flow cytometry and 200 nM Cy3-labeled siRNAs (Fig. 3).
Red and green populations represented cell counts of free siRNA and
NC-mediated delivery, respectively. RS-NCs showed fluorescence-positive
populations of 93.0, 81.5, 55.1, 32.5, and 21.3% in MDA-MB-231, BT-474,
MCF-7, SK-BR-3, and RAW 264.7 cells. On the other hand, RT-NCs showed
fluorescence-positive populations of 6.5, 4.6, 4.6, and 0.6% in MCF-7,
BT-474, MDA-MB-231, and SK-BR-3 cells. RT-NCs were not applied to RAW
264.7 cells due to low cellular uptake efficiency. For comparison,
R11-NCs showed fluorescence-positive populations of
20.3, 3.7, 2.0, 1.0, and 0.8% in MCF-7, BT-474, MDA-MB-231, RAW264.7,
and SK-BR-3 cells in high order. Resultantly, RS-NCs showed the best
cellular uptake efficiency. Therefore, RS-NC was chosen for further
studies.
MDA-MB-231 cellular uptake and internalization of NCs were observed
using fluorescence microscopy and Z-stack imaging (Fig. 4). Blue, green,
and red fluorescences represented the nucleus (Hoechst 33342), actin
filament (Phalloidin), and NCs (Cy3), respectively (Fig. 4A). RS-NCs are
represented as well-dispersed red dots (NCs) in the Cy3 image, and some
of the red dots entered into cells, and some of them remained outside of
cells in the merged image. RT-NCs are represented as some red dots in
the Cy3 image, and most red dots are located close to the cells in the
merged image. R11-NCs showed smaller dots than RT-NCs in
the Cy3 image, and most red dots are located close to the cells in the
merged image. On the other hand, only siRNAs did not show any red dots
in the Cy3 image. Next, the z-stack image indicated that RS-NCs are
located in MDA-MB-231 cells (Fig. 4B). Red and green fluorescence
represented actin filament (Cy3) and RS-NC (FITC). The z-stack
cross-section images of the x and y-axis white dotted line indicated
RS-NCs are located inside the cell.
The endocytosis pathway of RS-NCs into MDA-MB-231 cells was studied
using flow cytometry and endocytosis inhibitors (Fig. 5). Red and green
populations represented cell counts without endocytosis inhibitor and
with endocytosis inhibitor, respectively. As a result, chlorpromazine,
methyl-β-cyclodextrin, cytochalasin D, and filipin III inhibited RS-NC
endocytosis of 27%, 35%, 23%, and 19%, respectively. In addition,
chlorpromazine, methyl-β-cyclodextrin, cytochalasin D, and filipin III
were known inhibiting clathrin-mediated endocytosis, lipid raft-mediated
endocytosis, caveolin-mediated endocytosis, and micropinocytosis,
respectively.