3.1 Morphology of Electrospun PLA
SEM images of plain PLA nanofiber and other PLA nanofiber membranes with different concentrations of encapsulated paclitaxel were presented in Fig. 2. The result indicated that nanofibers of pure PLA and PLA incorporated with paclitaxel displayed uniform fiber size and contour length across the entire region of deposition area while the formation of spindles was negligible after the electrospinning procerss. Moreover, it was shown that plain PLA nanofibers possessed smooth surface morphology without the formation of detectable pores. To quantify the nanofibers’ dimension, the diameter of at least 15 nanofibers were randomly selected from each SEM image of membrane and were measured with image analysis software. The average diameters of nanofibers for three different membrane samples were summarized in Fig. 3. The result indicated that all samples displayed nanofibers with averaged diameters ranging from 0.36 – 0.43 mm.
3.2 Fourier-transform infrared spectroscopy (FTIR)
FTIR reveals the molecular features on polymeric thin-film through the determination of the unique vibrational modes of various chemical groups. FTIR spectra in the range of 500 – 3500 cm-1 for spin-coated PLA thin films mixed with different concentrations of paclitaxel (PLA+PTX50% and PLA+PTX100%) were shown in Fig. 4, with all absorption peaks fitted by Gaussian fitting in each sample’s spectrum. The FTIR spectra of plain PLA and pure paclitaxol were shown alongside as controls. In general, major FTIR peaks of plain PLA determined by Gaussian fitting included the following vibrational groups: C-O stretch at ~1087 cm-1, C-O-C at ~1183 cm-1, O=C-O in ester groups at ~1755 cm-1, O=C-O stretch in ester groups at ~1130 cm-1 and CH3 at ~1458 cm-1. The result as mentioned above corresponded well with all the basic chemical groups found along the backbone of PLA. For PTX, major vibrational modes included C-H out-of-plane or C-C=O deformation at 689 cm-1, C-H in-plane deformation at 803~941 cm-1, C-O stretching at 1049~1090 cm-1, C-N stretching at 1274 cm-1, CH3 deformation at 1330~1380 cm-1, C=C ring stretching at 1444 cm-1, C-C stretching at 1579~1652 cm-1, N-H bending at 1640 cm-1, C=O stretching at 1720~1727 cm-1, (C=O stretching) of amide group at 1733 cm-1, CH3/C-H stretching at 2541~2973 cm-1, -CH sp3 stretching at 3066 cm-1, N-H/O-H stretching at 3339 cm-1, agreed with those peaks of pure paclitaxel as reported in the literature [4, 6, 14-29]. Those main and minor vibrational modes of plain PLA and pure paclitaxel were summarized in Table 2.
3.3 Contact Angle Measruement
The averaged contact angles of PLA , PLA+PTX50% and PLA+PTX100% nanofiber membrane, supported on respective spun coated thin film were shown in Fig. 5. Regardless of the composition, the water contact angles of all samples were larger than 90°, implying high hydrophobicity displayed on these nanofibers. However, the incorporation of PTX into PLA matirx made the nanofiber coated substrate less hydrophobic as show by averaged water contact angles of 119.7° and 124.5° in PLA+PTX50% and PLA+PTX100%, respectively, compared to the higher average of of 139.8° in pure PLA.
3.4 Cell Culture
3.4.1. Biocompatibility of Pure PLA Nanofibers and Films
To ensure that PLA or PLA/PTX nanofiber membrane impose negligible cytotoxicity, each sample was immersed in plain culture medium for 72 hours which was subsequently extracted for culturing HCT-116 cells in-vitro. For the ease of direct comparison between the three types of nanofiber membranes, MTT data in terms of optical density ratio ( ) instead of individual MTT data. Fig. 6 showed the optical density ratio of HCT-116 cells determined from MTT assay at 48 hours after culturing in the three types of extracted liquid medium. The result indicated that the optical density ratio of HCT-116 cells incubated with extracted culture medium from glass, PLA thin film and PLA nanofiber membrane stays at around one.
3.4.2 Toxicity of PLA/Paclitaxel Mixed Nanofibers
Next, PLA or PLA/paclitaxel nanofiber membrane is immersed in liquid medium for the culture of HCT-116. Fig. 7 presented the optical density ratio from the MTT assay of HCT-116 cells which were cultured in the presence of PLA or PLA/PTX50% or PLA/PTX100% nanofiber membrane in liquid medium for 6 hours, 18 hours and 24 hours. The control group included HCT-116 cells cultured in liquid medium in the prescence of PLA nanofiber membrane. The optical density ratio of HCT-116 cells at each time point was determined as one for the control as mentioned above because there is an absence of paclitaxel in the liquid medium. After 6 hours of culture, the optical density ratio of HCT-116 cells was reduced by 73% and 81% in PLA/PTX50% or PLA/PTX100% containing medium, respectively, compared with that for cells in control group (with PLA nanofiber membrane). Similarly, the optical density ratio of HCT-116 cells was reduced by 78% and 81% in PLA/PTX50% or PLA/PTX100% containing medium, respectively, compared with that for cells in control group after 18 hours of cell culture. The result indicated that the prolonged release of paclitaxel was maintained by the PLA nanofiber carrier within 18 hours of drug adminstation. After 24 hours of cell culture, the optical density ratio of HCT-116 cells was reduced by 33% and 36% in PLA/PTX50% or PLA/PTX100% containing medium, respectively, compared with that for cells in control group after 24 hours of cell culture.
Fig. 8 showed a phase contrast image under 100X magnification of cultured HCT-116 in the presence of PLA or PLA/PTX50% nanofiber membrane in liquid medium after 24 hours of cell seeding. In the absence of paclitaxel in PLA nanofiber sample, HCT-116 cells populating at high density on the membrane surface displayed more elongated cell shpe corresponding to the normal morphology of this cell line as shown by the formation of membrane extensions. In contrast, most HCT-116 cells rounded up by transforming into a circular shape rather than the usual stretched and randomly stack up morphology (see the selected view) due to the loss of proliferative activities of the cancerous cells. At the same time, the cell density on the petri dish was significant reduced by PLA/PTX50% compared to that of PLA. The result indicated that paclitaxel acted as an effective toxic molecules presented to HCT-116 cells in the liquid medium before the cells were able to adhere on the nanofiber modified surfaces.
3.5 Cell Cycle
Fig. 9 showed the percentage of different phases within the cell cycle of HCT-116 cells after 24 hours of seeding in the liquid medium pre-incubated with PLA or PLA/PTX50% or PLA/PTX100% nanofiber membrane. In spite of the change in the concentrations of paclitaxel in the nanfiber membrane, HCT-116 in the G1 phase (~60%) overwhelms all others in other phases of cell cycle. Intuitively, G1 phase occurs when cells grow normally through the synthesis of various enzymes and nuitrients for getting ready for DNA replication in the S phase. The result indicated that most HCT-116 cells stay in G1 phase 24 hours after seeding without going into S phase through the G1 checkpoint. Secondly, around 20% of cells was at either S or G2/M phases cultured with liquid medium which was pre-incubated with either PLA or PLA/PTX50% or PLA/PTX100% nanofiber membrane.
Different concentrations of paclitaxel have different impacts on the apoptosis of cancer cells. Under a low concentration (<200 nM), the cell cycle may not be directly affected as paclitaxel may not be able to alter the overall architecture of the microtubules [10, 31-34]. The cell can still maneuver into prometaphase and arrested at the G2/M phase then followed by p34, cdc2 activation and Bcl-2 phosphorylation, leading to the eventual apoptosis [10, 33-39]. In our cases, the released paclitaxel concentration is estimated at around 32 – 68 nM (see Appendix) for the optical density ratio of 0.26 and 0.19 respectively. It could be concluded that the anticancer effect is attributed to the lower concentration of paclitaxel, which would have a direct impact on the microtubules of HCT-116, e.g., increased difficulty in passing thought the S1 checkpoint but not the overall mitosis [38-42].