Figure 3 . Confocal laser scanning micrographs of NPC43 cells incubated with PTX and PEG-PTX NPs for 16 h in 100×100 μm2 microwells. Tubulin in cells was stained by tubulin-tracker green (green fluorescence), and overlays of tubulin and bright field (BF) images.
For its therapeutic effect, PTX can promote the assembly and polymerization of tubulin, which will result in inhibition of mitosis and motility. Hence, after NPC43 cells were seeded in PDMS microwells and treated with PTX or PEG-PTX NPs for 16 h, the microtubules of NPC43 cells were marked by tubulin-tracker green assays. The concentrations of PTX and PEG-PTX NPs used were 5.85 nmol/mL for all subsequent experiments. As shown in Figure. 3 , NPC43 cells in 100×100 μm2 microwells without any treatment, as a control group, showed uniform and network-like green fluorescence, which exhibited that microtubules in the control group possessed well-organized and reticular distribution to maintain the stretched shape of cells. However, after the NPC43 cells were treated with PTX for 16 h, they became rounded, and microtubules were assembled together to show a bright and heterogeneous green fluorescence. For cells treated with PEG-PTX NPs for 16 h, bright green fluorescence of the tubulin bundles was observed because microtubules were gathered into large cluster but some cells still had elongated shape. In addition, the microtubules of NPC43 cells in 50×50 μm2 microwells and 150×150 μm2 microwells as shown in Supporting Figure. S1-S2 were also stained with green fluorescence. There was no difference for morphologic change of microtubules in microwells with different sizes. These results showed that PTX could be released from PEG-PTX NPs to act on intracellular microtubules, and PTX was more toxic than PEG-PTX NPs for NPC43 cells.
To further evaluate the effect of the microwell size and the degree of the confinement on the cytotoxicity of PTX and PEG-PTX NPs, the percentage of cell division and cell disruption were calculated as they represented the cellular activity and cell death. After NPC43 cells were seeded in the microwells and incubated with PTX or PEG-PTX NPs, a glass cover was placed on the top of the microwells to provide the additional confinement and limit the exchange of nutrients and drugs between the microwells and the outside surrounding. Six 3D platforms with 50×50, 100×100, and 150×150 μm2 microwells without and with the cover were used to evaluate the cytotoxicity of PTX and PEG-PTX NPs. At first, the effect of the cover on cell proliferation was presented by the percentage of cell division. As shown in Supporting Figure. S3, after NPC43 cells were incubated for 16 h without any drug treatment, cells could divide in all microwells without and with covers. Furthermore, the percentage of cell division was calculated as shown in Supporting Fig. S4 and Table S1, which exhibited that the cell division was inhibited in microwells with covers. But the size of microwells did not affect cell proliferation because the percentages of cell division were similar for 50×50, 100×100, and 150×150 μm2microwells without (~11%) and with (~7%) covers. There was no cell division after cells were treated with PTX or PEG-PTX NPs because drugs inhibit mitosis.