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.