2.1 Materials
Material Chemical
Formula
Purity Vendor
Anodic aluminum oxide Al2O3 Whatman Anodisc
13 (pore diameter:
Tetramethylammonium hydroxide C14H13NO 25 % Fluka
Sodium hydroxide NaOH 99 % Riedel-de Haёn
Absolute Ethanol C2H5OH Sigma-Aldrich
Magnet NeFeB Tesla Technology
HeLa cell T.K. Wu Lab.,
Hsinchu , Taiwan Sigma-Aldrich RPMI 1640 medium with folic acid
RPMI 1640 medium without folic acid
Invitrogen
DMEM medium with folic acid Sigma-Aldrich
DMEM medium without folic acid Sigma-Aldrich
Phosphate buffered saline
(3-(4,5-Dimethylthiazol-2-yl)-2,5-d iphenyltetrazolium bromide, a tetrazole), MTT
C18H16BrN5S Sigma-Aldrich
Dimethyl sulfoxide, DMSO C2H6OS 99.9 % J.T. Baker
Sodium azide NaN3 99.5 % Sigma-Aldrich
2.2 Instrument
Field Emission Scanning Electron Microscope (FESEM) Hitachi S-4000:
Accelerating voltage: 0.5 ~ 30 kV, Resolution: 1.5 nm at 25 keV Transmission Electron Microscope (TEM)
Temperature range at the sample space: 1.8 K ~ 350 K UV-Visible Spectrophotometer
2.3 Preparation of MCNTs composites 2.3.1 Template assisted synthesis of CNTs
Several of pieces of AAO templates were placed on a quartz boat and put into a tube furnace. The temperature was raised by a rate of 20 oC/min under 1 atm of Ar (flow rate: 10 sccm). When the temperature reached 550 or 650 oC, the carrier gas was closed and the reaction gas acetylene (C2H2) was passed into the quartz tube at a rate of 2.8 sccm. The acetylene gas was decomposed at 550 or 650 oC, while the deposition of carbon into the AAO template occurred. After 30 min, the color of AAO template turned black. The product CNTs inside the AAO channels were obtained.
2.3.2 Fe
3O
4solution preparation
For Fe3O4 solution preparation, iron (III) chloride hexahydrate (FeCl3‧6 (H2O), 99 %) (1 M) and iron (II) chloride tetrahydrate (FeCl2‧4 (H2O), > 98 %) (2 M) were prepared by dissolving the iron salts in an HCl solution (2 M), respectively.
2.3.3 Synthesis of MCNTs
Several CNT@AAO membranes were placed into a three-necked bottle (reaction process shown in Figure 2.1) and pumped below 10-2 torr for 5 min. FeCl3(aq)
(1 M, 4 mL) and FeCl2(aq) (2 M, 1 mL) solutions were added into the bottle under vacuum atmosphere. The three-necked bottle was slowly shaken for at least 5 min to allow the mixture solution to flow into the CNT@AAO membranes. The pump was closed and Ar gas passed into the three-necked bottle to prevent the Fe3O4
nanoparticles from further oxidation. Then, the remaining solution was removed after the mixing. A permanent magnet was placed under the three-necked bottle bottom.
The addition of NH4OH(aq) (4 M, 8 mL) lead to a black precipitate inside and on the CNT@AAO membranes. The solution color changed from brown to black. Then, a drop of tetramethylammonium hydroxide was added to the three-necked bottle to
produce a well dispersed Fe3O4 colloidal solution.25 After the three-necked bottle was slowly shaken, the remaining solution was removed. Deionized water was added to wash the CNT@AAO membranes. This procedure was repeated several times to remove excess ions and tetramethylammonium salt in the suspension. Finally, the washed CNT@AAO membranes were immersed in NaOH(aq) (5 M, 10 mL) at room temperature for three days to remove the AAO membrane. The MCNT was isolated by applying a permanent magnet and was washed several times to neutrality. The resulting product was dried in vacuum to offer MCNTs for further investigation.
Figure 2.1 MCNTs synthesis procedure.
2.4 Ex vitro measurement of heating of a MCNT solution by NIR radiation
MCNT solutions were prepared by sequential dilution in PBS buffer so that the final nanotube concentrations of 2, 1, 0.5 and 0.1 µg/µ L were obtained. They were irradiated using a 400 mW laser diode with a wavelength of 808 nm at a power density of 2.04 Wcm-2, a spot size of 0.5 cm diameter and the distance of laser head is about 2 cm from the bottom of well. Temperatures were measured in 30-s intervals with a thermocouple placed inside the solution for a total of 7 min. The thermocouple was placed away from the path of the laser beam to avoid direct exposure to the laser light. media respectively: the FA-free DMEM medium and the complete DMEM medium.
They were also supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin-streptomycin in 5 % CO2 and 95 % air at 37 oC in a humidified incubator.
Macrophages were cultured in complete DMEM medium supplemented with 10 %
overexpress FRs on cell surfaces. A549 and HeLa cells were passaged for at least four rounds in the FA-free medium before use to ensure overexpression of FR on the surface of the cells (FR+ cells). A549 and HeLa cells were prepared by culturing them in abundant FA medium to obtain FR- cells 14.
2.7 Uptake of MCNTs by cells
Firstly, the A549 and HeLa cells were cultured in two different media respectively: the FA-free medium and the complete medium supplemented with 10 % FBS and 1 % penicillin-streptomycin, followed by exposure to MCNT (1 µg/µ L, 30 µ L) in PBS buffer at 37 oC for 16 h. Finally, the incubated cells were washed with a
After the laser irradiation, the cells were rinsed with PBS buffer two times and then cell viability was assessed.
2.9 Optical microscopy
The cells were imaged by a Eclipse 80i Nikon microscope. Before the analysis, the cells were seeded in chambered coverslides or 96-well microplate for 24h and then incubated with MCNTs for 16 h. For staining cells, hematoxylin and eosin (H &
E) was added to each well and rinsed with PBS (1 mL) at room temperature before imaging.
2.10 Determination of cell cytotoxicity
The cell culture, laser radiation and cell cytotoxicity procedures were shown in Figure 2.3. Cell cytotoxicity assay was used to monitor cell viability with a
colorimetric tetrazolium salt-based assay. To determine the cytotoxicity of MCNTs, cancer cells were cultured in a 96-well microplate for one day and then incubated with different concentrations of MCNTs (A549: 1 to 3 µg/µ L, HeLa : 0.5 to 2.5 µg/µ L) for 16 h. After rinsed with PBS buffer (1 mL), a microculture tetrazolium (MTT) solution (300 µL) was added to each well to dye the cell. MTT was chemically reduced by cells into formazan. Then, after 4 - 6 h, a dimethylsulfoxide (DMSO) solution (200 µ L) was added into each well to dissolve formazan. The formazan concentration and optical absorbance at 560 nm with subtracting a background signal at 630 nm provided a measurement of the metabolically active live cells to determine cell viability. To detect photothermal cell viability, cancer cells were incubated without MCNTs for 48 h and then added an MCNTs solution (2 µg/µ L), followed by irradiation (808 nm laser, 2 W/cm2). Optical density at 560 nm with a subtraction of the signal at 630 nm (O.D 560), was read with a UV-Visible spectrophotometer to determine the viability of the cells.
Figure 2.3 Schematic representation of cell culture, laser radiation and cell cytotoxicity procedure.