The experimental setup for the study PLA/PTX nanofiber and thin films is laid out in Fig. 1. It needs to be noted that either nanofiber or thin films used herein is prepared from a mixture of PLA (40 wt. %) and paclitaxel. In general, nanofiber membrane are fabricated by electrospinning technique while thin film is formed by spin coating on a glass substrate. For material characterizations and biological studies, we place various samples of PLA/PTX nanofiber membrane on top of thin film of same composition which was spin-coated on a glass substrate for convenient measurements in various instrments.
2.1 Substrate and Materials
Glass coverslips (Corning 1737) were cut into a dimension of 10 mm ´10 mm by tungsten cutter and then ultrasonically cleaned in KOH (85%), followed by acetone (99.9%), DI water, and finally by alcohol (90%). Each cleaning step took around 10 minutes. After thorough cleaning, the glass substartes were blown to dry by nitrogen gas.
Poly(L-lactide) (, Synthesis C3H4O2]n, CAS 26161-42-2, FL-94829, Sigma-Aldrich, St. Louis, MO USA) made from lactide in the form of white to light yellow powder was dissolved in trifluoroacetic acid (TFA, 99%, CF3CO2H, , CAS 76-05-1, Alfa Aesar, Ward Hill, MA USA) and then continuously stirred for 24 hours at room temperature before used for electrospinning. The concentration of PLA solution is listed in Table 1. Paclitaxel (CAS: 33069-62-4, C47H51NO14) purchased from Sigma-Aldrich Inc. (UNI-ONWARD Corp. New Taipei City, Taiwan) in a powder form was dissolved in dimethyl sulfoxide (DMSO, CAS: 67-68-5, C2H6OS, Sigma-Aldrich, UNI-ONWARD Corp. New Taipei City, Taiwan). To obtain the best quality in the electrospun nanofibers, paclitaxel/DMSO solution (2.5mg/100μl (50%) or 5 mg/100μl (100%)) was mixed with PLA (40 wt. %) solution by using the volumetric ratio of PLA/paclitaxel = 3ml: 200μl to form the finalized polymer solution for either spin coating or electrospinning.
2.2 Spin Coating
First of all, glass substrates were treated by reactive-ion etching (RIE, 50w, Ar and O2 flow rates of 30sccm) for 30 min in order to increase the surface hydrophilicity. Then PLA/PTX thin film with various compostions was produced by spin coating (SWIENCO SP-02, Power Assist Instrument Scientific Corp. Taoyuan, Taiwan) on glass substrate in a solution mixture of PLA and PTX. The optimized spinning speed of 3000 rpm was set to from different types of PLA/paclitaxel thin films. All coating processes were completed within 4 min in order to allow the solution to cover across the entire glass substrate. After coating, we let the sample to dry for around 30 min in atmosphere environment before electrospinning of the polymeric nanofibers on top of the PLA/paclitaxel thin films.
2.3 Electrospinning of Nanofiber Membrane
The electrospinning of PLA/PTX was carried with commercially available electrospinning system (FES-COE, Falco, Taiwan) which consists of a syringe pump, a power supply and a two-dimensional motorized platform (collector) for the glass substrate. In brief, the solution mixture of PLA and PTX was first drawn into a glass syringe before the eventual depostion onto a substrate. The operational parameters of the electrospinning process are listed in Table 1. It must be noted that the distance between the syringe’s needle tip and substrate is optimized by trial and errors for depositing PLA/PTX nanofiber membrane with uniform thickness. The dimension (diameter) of nano-fibers is controlled by both applied voltage and flow rate. In general, the higher the applied voltage or flow rates, the thinner the fibers were produced. However, exceedingly high voltage often induces arching between the glass substrate and needle, which leads to the breakup of a streaming jet into droplets. Physical parameters in Table 1 were chosen after many rounds of process optimization in order to assure the highly reproducible properties of PLA/paclitaxel nanofibers.
2.4.1 Surface Morphology of Layer Film and Nanofibers
The diameters of electrospun PLA/paclitaxel fibers were measured from images by scanning electron microscope (SEM, S-3400N, Hitachi, Japan) with Image J software. For SEM measurement, the voltage of the accelerated electron beam is set at 15 kV and the magnification is chosen to be 2,000 X for the best resolution. The average and variances of the nanofiber diameters is averaged from 15-20 fibers ramdomly selected acoss the entire region of any SEM image.
2.4.2 Molecular Structure
To determine the vibrational modes of molecular bonding in the deposited PLA/paclitaxel layer films and nanofibers, Fourier transformed infrared spectrometer, FTIR (Perkin-Elmer Pentagon 1005, USA) equipped with He-Ne laser as excitation source at 632.8 nm (25 mW)) was used to detect the infrared absorption of the samples in the range of 450–4000 cm-1 with a resolution of 1 cm-1 in reflection mode. The FTIR spectrum of a plain glass substrate was first probed with FTIR and used as a control for comparing with those of other samples. Moreover, the IR detector is set at the backside of each sample. The spectrum was numerically analyzed with Fityk 0.9.8 software using Gaussian fitting to identify all peaks against the vibrational modes of various types of chemical bonds. Major FTIR absorption peaks of both PLA and paclitaxel from previous studies are listed in Table 2 [4, 6, 14-29].
2.4.3 Contact Angle Measurement
The contact angles of deposited films were measured from the image of water droplets (1 ml) on the surface of each sample as taken by a CCD camera (Watec, WAT-902B, Watec Corp, Japan). Images were further analyzed by Image J (ver. 1.52k released on 29 January 2019, National Institute of Health US) to obtain the contact angle.
2.5 Cancer Cell Culture
2.5.1 Cell Preparation
Human colorectal carcinoma cells (HCT-116) were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan) for our study. These cells were stored in Ependroff tubes and kept in -20oC freezer until use. Before each experiment, a batch of cells was first incubated and shaken in water bath at 37oC for about 30 minutes. After temperature equilibration, HCT-116 containign suspension is pipetted into the fresh culture medium (Dulbecco’s Modified Eagle Medium/High Glucose powder, GibcoÒ, Thermo Fisher Scientific, U.S.A.) inside a petri dish of 10 cm in diameter. Using the existing established protocol, we cultured HCT-116 in an temperature-controled incubator (ShelLab, 2424IR, USA) at 37oC with a supply of 5% CO2 and 95% relative humidity. The cells were typically cultured for 2 to 3 days before they were trypsinized and pipetted into clean Ependroff tubes. The entire suspension in Ependroff tubes was then centrifuged for 3 minutes at 1300 rpm. After centrifugation, the supernatant was discarded and cells containing sediment were respuspended in culture medium before loading into a 24-well plate for subsequent tests. The concentration of seeding cells was set at around 105 cells/500μl in order to avoid over-crowding or under-growing of cells in the subsequent culture. For cell culture study, all PLA/palitaxol nanofiber samples were sterialized by UV light for at least 15 minutes and then placed in a 24-well dish for all biological tests.
2.5.2 Cell Cycle Test
First, PLA/paclitaxel samples were loaded in a 6-well plate and submerged in fresh culture medium before placing inside the CO2 incubator. After 2 hour incubation, HCT-116 cells at a cell density of around 105 cells/500μl were loaded into the wells of 12-well plate. Followed by an additional round of incubation for 24 hours, the 12-well plates were taken out from incubator and the cell containing suspension were subjected to centrifugation in order to remove supernatant. Next, the cell concentration was reduced from 5x105 to 1x106 cells/500μl in every sample. The dilution process involves the addition of Solution 10 lysis buffer (acidic aqueous solution, ChemoMetec) mixed with DAPZ (volumetric ratio 1000:1) and Solution 11 stabilization buffer (basic aqueous solution, ChemoMetec). After the dilution, the stained cells were transferred to A8 slides (NC-Slide A8, ChemoMetec A/S Danmark), which were later analyzed by Chemometec NucleoCounterÒ (NC-3000, ChemoMetec A/S Danmark) for cell counting and cell cycles assay.
2.5.3 Cell Viability
MTT is a yellowish and water-soluble tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide) and is used for measuring viability of cells. In the presence of nicotinamide adenine dinucleotide (generated by active metabolism in cells), the tetrazolium dye contained in MTT is oxidized through the redcution of NADH to NAD+, leading to the formation of insoluble formazan with purple color. The insoluble formazan deposited on the cell surface was dissolved with isopropanol or other organic solvents. The dissolved solution has representative optical absorbance peaks at 562 and 650 nm identifiable by a spectrophotometer (Epoch 2, Microplate Spectrophotometer, BioTek Instruments Inc. USA), whose intensities are proportional to the concentration of formazan. As MTT is found in all living cells, its concentration can be used as a reliable marker for quantifing the number of viable cells. The average intensity of both absorption peaks is usually expressed as optical density (OD) which is defined as