Figure 7 . NPC43 cell morphology after various treatments. (a) Scanning electron micrographs of NPC43 cells with PTX and PEG-PTX NPs added over 16 h in 100×100 μm2 microwells without and with cover. Cell area after cells treated with PTX and PEG-PTX NPs for 16 h in microwells (b) without cover and (c) with cover. (d) Cell aspect ratio after NPC43 cells treated with PTX and PEG-PTX NPs for 16 h in 100×100 μm2 microwells without and with cover. Number of NPC43 cells counted is marked in white.
At last, the morphology of NPC43 cells in microwells was obtained by SEM. As shown in Figure. 7 and Supporting Figure. S12-13, for the NPC43 cells without any treatment in microwells without and with covers, they elongated and had many extended filopodia. However, the NPC43 cells became rounded and had few filopodia after treatment with PTX for 16 h. And the NPC43 cells treated with PEG-PTX NPs were elliptical with a few filopodia. To analyze the change of the cell morphology, the cell area and aspect ratio were evaluated as shown in Supporting Tables S4-S5. As shown in Figure. 7b, the NPC43 cells without any treatment possessed the largest cell area and the NPC43 cells treated with PTX had the smallest cell area. Cell area for the nPC43 cells treated with PEG-PTX NPs was slightly larger than the NPC43 cells treated with PTX. In microwells with covers, the cell area was about 200 μm2 as shown in Figure. 7c, which was smaller than the cell area of over 300 μm2 for the NPC43 cells in microwells without covers. Cell area was similar for the NPC43 cells treated with PTX and PEG-PTX NPs. In 100×100 μm2microwells, the cell area was 345±127 μm2 for control without covers, 115±39 μm2 for PTX without covers, 165±64 μm2 for PEG-PTX NPs without covers, 216±63 μm2 for control with covers, 139±44 μm2 for PTX with covers, and 135±44 μm2 for PEG-PTX NPs with covers. Furthermore, the aspect ratio of cells, which illustrated the cell elongation, was calculated. As shown in Figure. 7d, in 100×100 μm2microwells, the aspect ratios were 1.98±0.78 for control without covers, 1.26±0.28 for PTX without covers, 1.55±0.22 for PEG-PTX NPs without covers, 1.87±0.87 for control with covers, 1.27±0.17 for PTX with covers, and 1.36±0.23 for PEG-PTX NPs with covers. These results were consistent with the average speed dependence on the microwell size and the presence of the cover. For the control group without covers, the NPC43 cells moved normally and the cells elongated with the highest aspect ratio. After being treated with PTX, the NPC43 cells became rounded, and the aspect ratio was the lowest. Supporting Figure. S14 shows that in 50×50 and 150×150 μm2 microwells, similar results were obtained, independent of the size of the microwells.
3. Conclusion
In this work, 3D microwells without and with covers were fabricated to study the interplay of the cytotoxicity from PEG-PTX NPs and cell behaviors. First, stable PEG-PTX NPs with about 110 nm diameter were successfully synthesized to treat NPC43 cells. At short time, small molecule drug PTX was more effective to treat NPC43 cells than PEG-PTX NPs. After 64 h of treatment, the effect was similar according to results from the MTT assays. When the microwells had covers on the top, they provided additional confinement, and the cell division was slightly inhibited. In microwells with covers, more cell disruption was observed than those in microwells without covers after the NPC43 cells were treated with PEG-PTX NPs. In microwells without and with covers, more cell disruption was found when the NPC43 cells were treated with PTX than the NPC43 cells treated with PEG-PTX NPs. In addition, the cell migration speed decreased in microwells with covers. After cells were treated with PTX or PEG-PTX NPs, the cell migration speed further reduced. The effect of drug formations, microwells confinement, and size of microwells on cell behaviors including the cell migration speed, cell area, and cell elongation were studied in detail. In microwells without and with covers, after the NPC43 cells were treated with PTX, the migration speed was the lowest among other groups including control without any treatment, and NPC43 cells treated with PEG-PTX NPs. For cells treated with PEG-PTX NPs in microwells with covers, the average speed was lower than those in microwells without the cover, especially in 150×150 μm2 microwells. Furthermore, cell area decreased when the NPC43 cells were in microwells with covers. After cells were treated with PTX or PEG-PTX NPs, cell area further decreased, and their aspect ratio reduced. NPC43 cells became smaller and more rounded after treatment with PTX, while they had the elliptical shape after treatment with PEG-PTX NPs. This work provides a new approach to evaluate the relationship between nanomedicine, 3D confinement, and cell behaviors in vitro .
4. Experimental Section/Methods
Materials : Polydimethylsiloxane (PDMS) pre-polymer was purchased from Dow. Trichloro (1H , 1H , 2H , 2H -perfluorooctyl) silane (FOTS) was purchased from J&K. Tetrahydrofuran (THF) and dimethylformamide (DMF) were purchased from Sigma. Paclitaxel was purchased from Xi’an Haoxuan Biological Technology Co., Ltd. PEGylated (methoxypolyethylene glycol 2000) paclitaxel (PEG-PTX) was synthesized according to the previous literature.[48-50] Tubulin-tracker green and thiazolyl blue tetrazolium bromide (MTT) assays were purchased from Beyotime.
Characterization: The diameter and size distribution of PEG-PTX NPs were measured by dynamic light scattering (Zeta-sizer Nano, Malvern). The migration of cells, cell division, and cell disruption were recorded by a Nikon Eclipse upright microscope. The fluorescence of tubulin in cells was measured by a confocal laser scanning microscope (TCS SP5, Leica). The morphologies of microwells, nanoparticles, and cells were measured by a scanning electron microscope (SU5000 FE, Hitachi). The absorbance at 490 nm was measured by a Bio-Rad 680 microplate reader to calculate cell viability. The migration speed, migration trajectories, cell area, and aspect ratio of cells were calculated by Image J software. To calculate the aspect ratio of the cell shape, an ellipse was fitted over the cell with a short axisa and a long axis b , so that aspect ratio was obtained byb/a .
Fabrication of Microwell Arrays: PDMS microwells arrays were fabricated according to our previous work.[47] Si was used to fabricate the mold of microwell arrays. Microwells were patterned on Si wafer using photolithography with AZ6130 as the photoresist. 50 μm tall pillars in Si was etched in a deep reactive ion etching system. The etch conditions included repeated 7 s passivation cycle with 85 sccm C4F8, 600 W coil power at 20 mTorr, and 14 s etch cycle with 130 sccm SF6, 600 W coil power, 20 W platen power at 40 mTorr. 34 alternative cycles were applied to fabricate the 50 μm tall pillars. A monolayer of FOTS was coated on the Si mold as anti-stiction layer. The PDMS prepolymer with a 10:1 base to curing agent ratio was cast on the Si mold, spin coated at 1000 rpm for 1 min, and cured at 80 °C overnight. Replicated microwell arrays were then peeled off from the Si mold for further process.
Synthesis of PEG-PTX NPs: Similar method was reported in our previous work.[54-56] Briefly, PEG-PTX (40 mg) was dissolved in THF (5 mL), and dropwise added into water (10 mL) under stirring. After THF were completely evaporated, PEG-PTX NPs were collected.
Cell culture: NPC43 cells[57-60] were cultured in Roswell Park Memorial Institute 1× 1640 medium (Gibco) with 10% fetal bovine serum (FBS, Gibco), 0.2% 2 mM rock inhibitor Y-27632 (ENZO), and 1% antibiotic antimycotic (Gibco). NPC43 cells were maintained in incubator at 37 °C with 5% CO2. The medium was changed every two days and the cells were passaged when they reached 70% confluency.
Cell viability assays: The MTT assays against NPC43 cells were carried out. Similar experimental process was shown in our previous work.[61, 62] Briefly, PTX and PEG-PTX NPs were added into cell culture medium with different concentrations. NPC43 cells were incubated with PTX and PEG-PTX NPs for 16, 40, and 64 h.
Time-lapse imaging: Microwell platforms with different sizes were bonded to a confocal dish using O2 plasma. After twice sterilization with 70% ethanol, the confocal dish with platforms was put in a plasma system and treated with 5 min O2 plasma to form a hydrophilic surface. The O2 plasma conditions were 20 sccm flow rate, 100 mTorr chamber pressure, and 100 W RF power. The dish was maintained in 1× phosphate buffered saline (PBS) before cell seeding. A total number of 105 NPC43 cells were seeded in the dish for each experiment and maintained in cell culture medium for 8 h incubation. After that, the medium was changed to a mixture of 1:1 complete cell culture medium and CO2independent medium (Gibco) supplemented with 10% FBS, 1% antibiotic antimycotic, and 1% 100× GlutaMax (Gibco). Then PTX (5.85 nmol/mL) or PEG-PTX NPs (5.85 nmol/mL) was added. A Nikon Eclipse upright microscope was used to capture cell movements for 16 h at 5 min intervals.
Cell preparation for scanning electron microscopy: Cells on platforms were washed by PBS twice and fixed by 4% paraformaldehyde for 15 min. The cells were then treated by ethanol with concentration of 30%, 50%, 70%, 80%, 90%, 95%, and 100% for 5 min each, and then dried in a critical point dryer (EM CPD300, Leica) for 4 h. The dried samples were coated with a thin layer of Au to avoid charging and placed in a scanning electron microscope (SEM) for imaging.