Figure 4 . Comparison of NPC43 cell disruption ratio with different treatments. (a) Optical images of NPC43 cells incubated with PTX and PEG-PTX NPs for 16 h in 100×100 μm2 microwells without and with glass cover. Cell disruption was highlighted in red circles. Percentage of cell disruption after PTX and PEG-PTX NPs addition for 16 h in different microwells (b) without cover and (c) with cover. Number of NPC43 cells counted is marked in white.
To study the cytotoxicity of PTX and PEG-PTX NPs, the percentage of cell disruption was calculated as shown in Supporting Table S2. As shown inFigure. 4a and Supporting Figure. S5-S6, some cells ruptured after NPC43 cells were treated with PTX or PEG-PTX NPs for 16 h in microwells with different sizes. As shown in Figure. 4b-c, the size of microwells had no effect on cell division. Therefore 100×100 μm2 microwells were used for further explanation. In 100×100 μm2 microwells without a cover, the percentages of cell disruption after PTX and PEG-PTX NPs addition for 16 h were 14.01±3.66% and 10.77±3.92%, respectively. However, the percentage of cell disruption was only 4.42±3.50% for cells without any treatment. This result showed that PTX was more effective to treat NPC43 cells than PEG-PTX NPs, which was consistent with the result from the MTT assays. Furthermore, in 100×100 μm2 microwells with a cover, the percentages of cell disruption after PTX and PEG-PTX NPs addition for 16 h were 13.12±3.44 and 12.23±5.26%, respectively. After NPC43 cells were treated with PEG-PTX NPs for 16 h, slightly more cells reptured in microwells with a cover compared to microwells without a cover. In the presence of PEG-PTX NPs, the additional confinement by the cover led to an increase in cell disruption. In addition, similar results were obtained for 50×50 and 150×150 μm2microwells.

2.3. NPC43 cell behaviors with PTX and PEG-PTX NPs treatments

Because cell migration speed is a key indicator for cell behaviors and PTX could inhibit cell motility, cell migration speed was monitored after cells were treated with PTX and PEG-PTX NPs in different microwells. Additionally, cells on flat surface were also tested for comparison. As shown in Supporting Figure. S7, for cells without any treatment, the migration speed of NPC43 cells decreased a little in 16 h. However, after NPC43 cells were incubated with PTX, the migration speed decreased to 0.1 μm/min after PTX treatment for 5.4 h. The time when the cell migration speed was equal to 0.1 μm/min was defined as t0.1. NPC43 cells moved slower after treated with PTX for 5.4 h because small molecule PTX could act directly on tubulin to inhibit cell movement. However, for cells treated with PEG-PTX NPs, t0.1 was 12.4 h which demonstrated that PEG-PTX NPs could continuously release PTX to slow down the NPC43 cell motility.