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.