3.1. Characterization of plain PCL and styrax loaded PCL nanofibers
The FTIR spectroscopy presented in Figure 2A was employed to reveal the presence of styrax in the structure of PCL nanofibers and the effect of different amounts of styrax on the chemical characteristics of nanofibers. When the spectra of electrospun membranes prepared at different proportions of content are examined, it was seen that there was no significant change in the basic functional groups of PCL after being loaded with styrax. The nanofibrous membranes exhibited characteristic peaks of PCL located at 2940, 2865 and 1722 cm−1 attributed to the stretchings of –CH3, –CH2 and C=O, respectively [34, 35]. In addition, the spectra showed the other absorption bands of PCL located at 1296 (C-O stretching), 1245 (asymmetric C-O-C strecthing) and 1190 cm−1 (asymmetric O-C-O strecthing) [36, 37]. Although these signals were displayed by all samples and no changes in the positions of these peaks were noted (including the spectrum of PCL-styrax composites), a significant increase in the intensity of the peaks was observed with the increasing amount of styrax. Nevertheless, a new peak around 1640 cm-1 corresponding to the double bond stretching vibration of C=C group of styrene, the main component of styrax shown in Figure2B, was observed in composites with high rates of styrax (PCL50% and PCL100%) [20, 38]. These changes show that styrax is successfully dispersed in nanofiber composites.
SEM was performed for accounting any difference in morphology of the PCL nanofiber blends with different amounts of styrax liquidus as compared to neat PCL fibers. With this approach, the morphological changes and size distribution histograms of the samples are presented in Figure 3 and Figure 4, respectively. All nanofibrous scaffolds had uniform and bead-free network structure with good continuity and smoothness. The morphological evaluation indicated that the structure of the electrospun nanofibers depended on the balsam concentration. As the weight ratio of balsam based on the polymer weight increased to 25, 50 and 100%, nanofibers started to be heterogeneous in structure. On the other hand, the mean fiber diameter obviously increased with the addition of styrax in the recipe. The average diameter for neat PCL fibers was calculated to be ~188.75 ± 44.54 nm. The styrax loaded hybrid fibers showed the diameters of around 192.70 ± 58.33, 316.11 ± 102.29, and 371.27 ± 108.61 nm for PCL25%, PCL50%, and PCL100%, respectively. These differences in mean fiber diameter may be attributed to the encapsulation of styrax in PCL fibers. According to this change, when the high-magnification SEM images of high balsam content nanofibers examined, the styrax loaded capsules indicated by yellow arrows can clearly be seen in Figures 3G and 3H. This similar structural change occurring in fiber morphology were also observed in the Hypericum perforatum oil-loaded fibrous membranes developed by Eğri and Erdemir [39].
Hydrophilicity and wettability of the membranes which are the important features for wound dressings were evaluated by determining the contact angle and water uptake capacity, respectively. From Figure 5A, the contact angle of all samples is found to be 90°, showing the hydrophobic nature of the nanofibrous scaffolds. The pristine PCL mat is more hydrophobic (127.07±1.03°) than the PCL25%, PCL50% and PCL100% samples and their contact angle are 124.02±0.69, 118.01±0.73 and 113.66±2.38, respectively. In different research articles, similar to hydrophobic nature of PCL, polyurethane-turmeric oil [40], and polyurethane-grape seed oil/honey/propolis incorporations resulted contact angle decrease compared with pure polyurethane scaffold [41]. The change in hydrophilicity as a result of adding a plant-based oil to the PCL synthetic polymer was evaluated in the study conducted by Ünalan et al. and they showed that loading of increasing amounts of peppermint essential oil on electrospun PCL fiber mats decreased the contact angle of the plain PCL fibrous mat [42].
On the other hand, to determine the water uptake ability of the scaffolds, gravimetric measurements were performed after immersion of samples in distilled water until the equilibrium water uptake capacity is reached. According to the results shown in Figure 5B, the water uptake capacity of high amounts of styrax liquidus incorporated nanofibers (PCL50% and PCL100%) was significantly different than the neat PCL fibrous sample. This result is dependent on the hydrophilic/hydrophobic nature of the materials as well as on the morphological properties of the nanofibers [43]. As we mentioned above, the surface of membranes have acquired a little hydrophilic property with the addition of styrax liquidus and consequently, their water uptake capacity may have increased. Additionally, the increase in pore size (Figure 3) with the addition of styrax may have also affected the swelling capacity because the high porosity can be defined as the presence of more free space available for water uptake [44].
3.2. DPPH Radical Scavenging Activity
It is generally known that the wound healing properties of plant derivatives are significantly associated with their antioxidant activities. Therefore, the antioxidant activity determined by measuring the DPPH radical scavenging activity of PCL and styrax loaded PCL (PCL25%, PCL50% and PCL100%) nanofibers was evaluated and the results were represented in Figure 6A. It was shown that plain PCL and different amounts of styrax loaded PCL nanofibers exhibited well antioxidant activity in comparison with the positive controls trolox and ascorbic acid. When the concentration of nanofibers was increased from 25 mg/L to 500 mg/L, the DPPH radical scavenging activities were increased from 26.4% to 71.4%, from 33.6% to 78.5%, from 54.4% to 82.7%, and from 63.5% to 86.7%, respectively. PCL100% sample was found to has a more effective DPPH radical scavenging ability at all concentrations. According to the results, radical scavenging potencies were concentration-dependent. It was observed that the antioxidant effect increased as the proportion of the styrax in PCL nanofibrous samples increased. The results are compatible with the literature [45]. Furthermore, this study results indicated that the styrax-PCL hybrid nanofibrous membrane could be used as a potential antioxidant biomaterial for future wound healing applications.
3.3. Metal chelating activity
Ferrous ion chelating activities of plain PCL, and PCL%25, PCL%50, and PCL%100 samples were studied by the ferrozine test. In the presence of chelating agents, the formation of ferrous-ferrozine complex is inhibited so that the magenta color of the complex is bleach. The results of the test prove that all samples exhibited chelating activities as seen in Figure 6B, chelating activities also were concentration-dependent. The ferrous ion chelating activities at 100 mg/L were 50.7%, 55.2%, 59.0%, and 63.0%, for PCL, PCL25%, PCL50%, and PCL100%, respectively. According to method applied tested nanofibers also exhibited chelating ability in the order of PCL100% (71.7%) > PCL50% (66.6%) > PCL25% (63.7%) > PCL (57.9%) at concentration of 200 mg/L. Metal ion chelating capacity is important since it reduces the concentration of the transition metal that catalyzes lipid peroxidation [46]. The metal chelating capacity of the styrax in PCL nanofiber is not good as the standard EDTA, but the decrease in concentration-dependent color formation in the presence of nanofibrous membranes indicates that it can be used as a metal chelator.
3.4. Biofilm inhibition activity
Biofilm formation as a result of bacterial infections is one of the most important and undesirable parameters that delay wound healing [47]. Biofilm-forming cells exhibit greater resistance to antibiotics, biocides, and extreme conditions than planktonic cells. In addition, biofilm cells are more difficult to remove or inactivate [48, 49]. In this method, the effect of PCL, PCL25%, PCL50% and PCL100% at 250 mg/L and 500 mg/L concentrations against the P. aeruginosa and S. aureus biofilm formed on polystyrene microplates was estimated by measuring the population of viable cells. P. aeruginosa and S. aureus are bacteria colonizing around the chronic wounds [47]. As shown in Figure 6C, when the concentration of PCL%0, PCL%25, PCL%50, and PCL%100 was increased from 250 mg/L to 500 mg/L, the biofilm inhibition activities against P. aeruginosa were increased from 59.4% to 68.3%, from 76.5% to 78.9%, from 83.0% to 85.0%, and from 86.5% to 90.6%, respectively. Similar results were obtained against the S. aureus and PCL100% showed the highest activity (93.3%) at 500 mg/L concentration. Obtained results prove that styrax in PCL nanofibers have the potential to be applied as an antibacterial and anti-biofilm agent to combat P. aeruginosa and S. aureus which are the most commonly encountered bacterial species in infected chronic wounds.
3.5. DNA cleavage activity
To determine the functionality of neat PCL and different amounts of styrax incorporated PCL nanofibers as DNA cleavage agents, the materials were investigated using supercoiled pBR322 plasmid DNA. The efficiency of the samples was tested by agarose gel electrophoresis. The results of the study are depicted in Figure 6D. At 250 μg/mL concentration, neat PCL did not exhibit cleavage activity (lane 2). Whereas, PCL25%, PCL50%, and PCL100% demonstrated single-strand cleaved DNA activities in lane 3, lane 4, and lane 5, respectively. At 500 μg/mL concentration, PCL25%, PCL50%, and PCL100% successfully cleaved DNA and double-strand DNA occured in lane 7, lane 8, and lane 9, respectively. Generally, the results suggest that Liquidambar orientalis Miller in PCL nanofibers have the potential as functional materials for medicine industries after further studies.
3.6. Antimicrobial activities
In vitro antimicrobial potentials of PCL, PCL25%, PCL50% and PCL100% were scrutinized in terms of minimum inhibition concentration (MIC) value against Gram-positive, Gram-negative bacteria and a fungal strain. MIC values obtained from the study are presented in Table 1. As can be seen, PCL and PCL25% were exhibited weak antimicrobial activity against all tested microorganism. Whereas PCL50% and PCL100% were shown well antimicrobial activity ranging from 16 to 64 μg/mL against tested Gram-negative bacteria E. coli, L. pneumophila, and P. aeruginosa. In addition to this, they were exhibited moderate antimicrobial activity ranging from 16 to 128 μg/mL against tested Gram-positive bacteria B. cereus, S. aureus, E. hirae, and fungus C. albicans. The neat PCL nanofibers did not display significant antibacterial activity. Similarly, a novel active film made of PCL containing sage extract was developed by Salevic et al. and they reported that plain PCL has no antibacterial effect [50]. In our study, styrax in PCL nanofiber samples (PCL 50% and PCL 100%) showed good antimicrobial activity. These results are in agreement with the other studies [19, 21]. The mechanism of biocidal action of plant-derived compounds is based on the degradation of the cell wall, damage to cytoplasmic membrane and membrane proteins, leakage of content out of the cell, and coagulation of the cytoplasm [50, 51]. The different antimicrobial activity of our samples against microorganisms can be attributed to the differences in the cell wall structure of Gram-positive and Gram-negative bacteria. According to our results, styrax loaded PCL nanofibrous membranes can be used as antimicrobial materials.
Table 1. The minimum inhibition concentration of tested microorganisms
Bacteria
|
Series*
|
PCL
|
PCL25%
|
PCL50%
|
PCL100%
|
E. coli
|
128
|
64
|
32
|
16
|
B. cereus
|
256
|
256
|
128
|
128
|
Legionella
|
128
|
64
|
32
|
16
|
S. aureus
|
128
|
64
|
32
|
16
|
P. aeruginosa
|
256
|
128
|
64
|
32
|
E. hirae
|
256
|
128
|
64
|
64
|
C. albicans
|
512
|
256
|
128
|
64
|
*mg/L
3.7. Cell Viability
In vitro cellular response to PCL100% nanofibers is presented in Figure 7. Direct test shows that PCL100% nanofiber scaffolds showed better cell viability compared to the control group. As it can be seen, there is significant difference between the cell viability of control group and PCL100% nanofiber samples at different days (Figure 7A). The PCL100% nanofiber showed cell proliferation rate 65.76 ±0.62%, 299.25 ±2.2%, and 497.48 ±0.37%, at 1, 3 and 5 day, respectively. The PCL100% nanofiber showed better cell proliferation rates than the control group. It was reported that the membrane with styrax enhanced adhesion and proliferation of MEF cells. The addition of styrax increased the hydrophilic nature of the pristine PCL membrane which might have favoured the enhanced MEF cell viability. In addition to this, styrax includes also active compounds such as; terpinen-4-ol, α-terpineol, sabinene, α-pinene , veridiflorol and p-cymene which are known to promote cell proliferation and have an antimicrobial activity on bacteria and fungi [20, 52]. However, the indirect cell proliferation rates for control group (standard medium) were higher compared to the PCL100% nanofiber (medium with PCL100% nanofiber degradation products) (Figure 7B). Such a behaviour has been reported before and needs further investigations. This might be due to the long term incubation of PCL100% nanofiber in medium (7 day) as described in different study [53]. Various investigations presents that PCL scaffold incubated one or three day in medium and results show that cell viability value was lower than the control group with this medium [54, 55].