Çörekotu Yağı İçeren Poliüretan Nanolifli Yüzeylerin Morfolojik Özellikleri Üzerine Bazı Proses Parametrelerinin Etkilerinin Araştırılması

Corekotu tohumu ve ondan elde edilen ekstraktlar antik caglardan beri alternatif tip alanlarinda uygulanmaktadir. Bu calismada, termoplastik poliuretan polimeri icerisine farkli konsantrasyonlarda corek otu yagi ilave edilerek, elektro cekim yontemi ile yara ortusu amacli nanolifli kompozit yuzeyler uretilmesi ve karakterize edilmesi amaclanmistir. Uretim esnasinda besleme unitesi ile toplayici arasindaki mesafe ve besleme orani degistirilerek; elde edilen yuzeylerin morfolojileri taramali elektron mikroskopu (SEM) ile analiz edilmistir. Nanolifli yuzeylerin, nanolif capi, gozenek boyutu ve gozeneklilikleri tayin edilmistir. Nanolifli yapi icerisindeki corek otu yaginin varligi FTIR analizi ile ve yuzeylerin islanabilirlik seviyesi temas acisi olcumleri ile incelenmistir. Sonuc olarak; surekli, uniform ve boncuk icermeyen nanolifler elde etmek icin corekotu yagi katkisi icin en uygun konsantrasyon ve optimum elektro cekim proses parametreleri belirlenmistir.


INTRODUCTION
Nigella Sativa (NS), commonly known as -black seed‖, is an herbaceous plant that grows around of Mediterranean countries (Zaoui et al., 2002). The black seed and its extracts have the wide variety of applications in the traditional medicine and it can be use on treatment of various diseases including diabetes, asthma, flatulence, polio, kidney stones, and abdominal pains. Many pharmacological studies have been conducted to investigate its biological activities and therapeutic properties such as analgesic and anti-inflammatory effect (Hajhashemi et (Burits and Bucar, 2000). Main components of black seed oil (BSO) are saturated/unsatured fixed oil, essential oil (0.4-0.45%), carbohydrate (33-34%), protein (16)(17)(18)(19).9%), amino acid, alkaloid, tannins, saponins, minerals (calcium, zinc, phosphate), vitamins (ascorbic acid, thiamine, niacin, pyridoxine and folic acid) (Güzelsoy et al., 2018). The thymoquinone, which is known to cause for many therapeutic properties, is a major active chemical component (30%-48%) of essential oil of the black seed (Ahmad et al., 2013). Many studies have been also issued to prove therapeutic effects of thymoquinone (Kalhori et al., 2018;Majdalawieh, 2017).
Polymeric nanofibers possess unique properties, such as ultra-fine fiber diameter, high surface area per unit mass, high porosity, small pore size, high mechanical properties, and extreme flexibility depending on the polymer structure. Therefore, they are favorable for many applications (Akduman and Kumabasar, 2017 andMi et al., 2015). Electrospinning is an effective method for the production of polymer nanofibers with the usage of electrostatical forces. In this method, a droplet of a polymer solution is subjected to an electrical field. With the increasing voltage, the solvent evaporates, leaving behind a mat of dry polymeric nanofibers on the collector (Tijing et al., 2012;Almetwally et al., 2017).
Due to their outstanding properties, nanofibrous mats present a wide range of application possibilities from agriculture to protective clothing. Among many application areas, nanofibrous mats are promising for medical applications such as tissue engineering, drug delivery, wound healing, tumor therapy, and barrier textiles due to their small fiber diameter, high surface area to volume ratio, small pore size, high porosity and high mechanical properties (Huang, et al., 2003;Chronakis, 2005).
Although electrospinning is relatively a simple method compared to other nanofiber production methods, there are many parameters affecting the morphology of the nanofibers. These parameters are mainly solution parameters (viscosity, surface tension, pH, molecular weight of the polymer, etc.), process parameters (flow rate, spinning distance, voltage, needle diameter, etc.) and ambient parameters (temperature and humidity). In order to produce an appropriate nanofibrous mat with desired properties, these parameters should be carefully selected (Teo and Ramakrishna, 2006;Theron et al., 2004).
Polyurethane (PU) is a highly used polymer in medical applications because of their biocompatible nature, non-toxicity, controlled degradation, good barrier properties and capability of gas and water permeability (Detta et al.,2010). They are a type of polyurethane (PU) composed of chemical reaction between isocyanates (hard segments) and polyols (soft segments) and contain repeating urethane groups (Sharmin and Zafar, 2012). They also have high elongation, good mechanical properties, water insolubility, abrasion and tear resistance properties The aim of this study is to produce PU/BSO nanofibrous mats by electrospinning, to determine the optimum solution and process parameters (flow rate and spinning distance) for the electrospinning of PU/black seed oil nanofibers, and to investigate the effect of solution properties and process parameters on the morphology of PU/BSO nanofibrous mats. Therefore, PU/BSO solutions with different concentrations were prepared. The solutions were characterized in terms of viscosity, pH and surface tension. PU/BSO nanofibers were produced by using single needle electrospinning process with different flow rates and spinning distances. The surface properties were investigated by Scanning Electron Microscopy (SEM) studies and contact angle measurements. Fourier Transform Infrared spectrometry (FTIR) analyses were performed in order to understand the chemical structure of the fibers. SEM studies showed that PU and PU/BSO nanofibers were successfully with diameters mostly below 500 nm. The diameters and contact angle values were slightly decreased depending on the BSO content in the nanofibrous mat. It was also seen that; process parameters have significant effect on the mat morphologies.

Materials
To prepare PU and PU/BSO electrospinning solutions, polyester-based thermoplastic PU (BASF Elastollan®) with a molecular weight of 107.010 g/mol and a bulk density of 1.19 g/cm 3 was used. Cold pressed black seed oil was supplied from a local farmer in Bursa-Mustafakemalpaşa (Turkey). Dimethylformamide (DMF) (Sigma-Aldrich, USA) was used as solvent to prepare the spinning solutions. All the chemicals were used as received without any further purification.

Production of PU and PU/BSO nanofibrous mats
Prior to the solvent preparation, the fatty acid composition of black seed oil was determined. Analysis of fatty acid composition of black seed oil was carried out using an Agilent 6890 N GC gas chromatography-flame ionization dedector (GC-FID) (Agilent Technologies, Palo Alto, CA). An HP-88 fused-silica capillary column 100m×0.25 i.d., with 0.20 um film thickness was employed. The injector temperature was set at 250 C. The GC oven temperature was held at 120 C for 1 min, then increased to 175 C at a heating rate of 10 C min −1 , 210 C at a heating rate of 3 C min −1 , 240 C at a heating rate of 5 C min −1 and the temperature was held for 5min. Standard test method of TS EN ISO 12966-2 and TS EN ISO 12966-4 was used in analysis. The test was repeated three times.
In this study, three different solutions were prepared with 5%, 10%, and 15% (v/v) BSO addition into the spinning solution. PU solutions without addition of BSO were also prepared as reference. In order to prepare PU/BSO electrospinning solutions, 10% (w/v) PU and BSO with different concentrations were dissolved in DMF and stirred at room temperature for 24 h. For the sake of brevity, the reference sample was denoted as PU, while the solutions with different BSO concentrations were denoted as PU/BSO5, PU/BSO10, and PU/BSO15.
The prepared solutions and pure black seed oil were characterized in terms of viscosity, surface tension, and pH. The viscosities of all solutions were determined by Brookefield viscometer at 100 rpm. Surface tension values of the solutions were measured by KSV-The Modular CAM 200 tensiometer and the pH values were determined by a Hanna pH tester.
Electrospinning was performed with an Inovenso Electrospinning Equipment. All experiments were carried out 15 kV at room temperature. The nanofibrous mats were collected on a rotating drum with a speed of 200 rpm. All electrospun mats were produced from electrospinning solutions of 20 mL. The details of the process are given in Table 1.

SEM studies
The surface morphologies of the nanofibrous mats were examined by a Scanning Electron Microscope (SEM) (Carl Zeiss Evo 40). Fiber uniformity, fiber diameter distribution, fiber evenness in the mats was observed. The surfaces were coated with a thin conducted gold layer prior to SEM observations. Fiber diameters were calculated from the SEM images by measuring 100 random diameters using image analysis software (Image J).

Pore Size and Porosity Calculations
In order to calculate pore size and porosity, the samples were cut as 2x2 cm 2 . Afterwards the samples were weighted by a digital scale and the thicknesses of the samples were measured by a digital micrometer. The measurements were repeated three times. The mean effective pore diameter (pore size) and porosity of the electrospun nanofibrous mats were calculated using the following equations (Zhu et Where ε is the porosity of the electrospun nanofibrous mat, ρe and ρf are the electrospun nanofibrous mat density and bulk PU density, respectively. D= πd/4(1-ε) ……………….…………………………………(2) Where D is the mean effective pore diameter, d is the mean nanofiber diameter and ε is the mean porosity of the sample.

Contact Angle Measurements
The contact angle values of the electrospun nanofibrous mats were determined using KSV-The Modular CAM 200 Contact Angle Measurement System. A water droplet was dispersed on each sample by a micropipette, and the images were captured as quickly as possible after water droplet was placed onto the sample surface. Three measurements were performed on each sample and mean values were recorded.

ATR-FTIR analysis
A Perkin-Elmer Spectrum 100 FTIR Spectrometer (Perkin Elmer, Waltham, MA, USA) equipped with an attenuated total reflectance (ATR) apparatus was used to obtain FTIR spectra of oil, PU and PU/BSO nanofibrous mats within the range of 600-4000 cm -1 with the resolution of 2 cm -1 .

Characterization of PU, BSO and PU/BSO solutions
Solution parameters play a key role on the morphology of the nanofibers. Table 2 shows the properties of the BSO, PU and PU/BSO solutions.
Viscosity of a solution is dependent on the chain entanglements in the solution. In electrospinning, a critical chain entanglement is required to obtain nanofibers (Mi et al., , Ramakrishna et al., 2005. It can be seen that, BSO addition into the spinning solution resulted in higher viscosities due to the increase in polymer concentration per unit area at a constant volume. Surface tension of the polymer solution directly affects polymer jet formation, as the electrostatic forces in the electric field must overcome the surface tension of the solution to form a polymer jet (Teo and Ramakrishna, 2006). With the BSO addition into the spinning solution, the surface tension values of the solutions were slightly increased indicating that higher electrical forces are needed to overcome the surface tension of the polymer droplet at the spinneret (Demir et al., 2001). This result is also compatible with the viscosity increase of the solutions.
Since pure BSO has an acidic nature, the pH of the PU/BSO solutions have slightly decreased, compared to pure PU solution. Adding an acidic component into the spinning solution leads to increase in the conductivity of the solution because of the increase in the number of ions. This may affect the diameter of the nanofibers. Thinner nanofibers can be produced from the more conductive solutions (Ramakrishna et al., 2005).

Characterization of PU and PU/BSO nanofibrous mats
The nanofibrous mats (Figure 1) were successfully produced from solutions of PU and PU/BSO by electrospinning. The properties of the electrospun nanofibrous mats were given in Table 3. Generally, PU nanofibers without BSO had diameters between 400-700 nm. The increase in viscosity of the solution leads an increase in diameter (Ramakrishna et al., 2005).With the addition of BSO, the viscosities of the solutions were increased. When the concentration of BSO in the solution was increased, the fiber diameters increased. However, PU/BSO nanofibers showed smaller diameters than PU nanofibers. This is maybe due to the pH change in the spinning solution. Since the number of ions increased with the addition of BSO, the diameters were decreased. In the study, the effect of flow rate and the distance between the needle and the collector on surface morphology were also investigated. When the flow rate was increased from 0.5 mL/h to 0.7 mL/h, nanofibers with larger diameters were produced (Figure 2), as expected. Also, with the increase in flow rate, the pore sizes of the nanofibrous mats were increased, nanofibers were flattened, and nanofiber web formation was observed. In electrospinning, if the flow rate is high, due to the greater volume of solution drawn from the needle tip, the jet needs longer time to dry. As a result, the solvents in the deposited fibers may not have enough time to evaporate. Evaporation of some amount of residual solvent from the fibers on the collector may cause the fibers to fuse together at contact points where they become flattened and form webs (Ramakrishna et al., 2005;Li andWang, 2013, Baji et al., 2010). Although the increase of the fiber diameter is caused by flattening, the increase in the pore size might be associated with the dissolution of some fiber during solvent evaporation and the formation of new pores.

Figure 1: SEM images of the nanofibrous mats
The distance between the needle and the collector also affect the nanofiber morphology and diameter. If the distance is short, the fiber cannot find enough time to solidify before reaching the collector. When the distance is increased, the polymer jet exposed to higher electrical forces, the flight time of the jet increases as a result, thinner nanofibers are obtained (Li and Wang, 2013). In this study, PU and PU/BSO nanofibrous mats were produced with different distances at 18 cm and 20 cm. It was seen that with the increasing distance, the nanofibrous mats with higher porosity, higher pore size, and thinner fibers were produced (Figure 3).  Contact angle measurements give information about the surface characteristics of the materials such as, surface wettability, adhesion, hydrophobicity, absorption, etc. (Hsieh, 2001). In this study, all PU and PU/BSO nanofibrous mats showed contact angle values higher than 90° indicating that all the surfaces were hydrophobic due to the chemical structure of PU polymer. Pure PU nanofibrous mats produced with different flow rates and distances had contact angle values changing from 103.5° to 118.4°. The lowest contact angle was observed as 86.4° for the samples produced from the solutions containing 15% wt. BSO. Although, BSO has a hydrophobic nature, adding BSO into the nanofiber structure decreased the contact angle values. Contact angle values are dependent on the compounds present at the surface, the surface roughness, and the surface morphology. Surface morphology and roughness of the nanofibrous mats are affected by the diameter of the nanofibers, pore sizes and porosity of the nanofibrous mats. Highly porous surfaces show high contact angle due to the air pockets trapped under the liquid drop (Darmanin and Guittard, 2014). In the present study, pure PU nanofibrous mats had higher porosity compared to the PU/BSO nanofibrous mats (Table 3). Therefore, more porous surfaces produced from pure PU solutions, showed higher contact angle values. This proved that surface structure has a significant effect on the wettability of the nanofibrous mats.‖. FTIR analysis were performed in order to show the presence of BSO in the nanofibrous mats ( Figure  4). FTIR analysis are not affected by the concentration of the spinning solution. Although, the concentration may affect the intensity of the peak, it doesn't affect the location of the peak. Therefore, FTIR spectrums of BSO, pure PU nanofibrous mat and PU/BSO nanofibrous mat were given in this study. The spectrum of pure PU exhibited peaks at 3327 cm -1 , 2954 cm -1 , 2865 cm -1 , 1727 cm -1 , 1701 cm -1 , 1597 cm -1 , 1527 cm -1 , 1220 cm -1 , 1142 cm -1 and 770cm -1 . The peak at 3327 cm -1 is a characteristic broad of PU that corresponds with stretching vibration of the N-H in urethane groups and the peak at 1597 cm -1 and 1527 cm -1 shows the vibration of the NH stretch (Tang and Gao, 2017). The other absorption peaks of pure PU were observed at 2954 cm -1 and 2865 cm -1 that denote CH 2 asymmetric vibration and CH 3 symmetric vibration, respectively (Tijing et al., 2012). The peaks at 1701 cm -1 and 1727 cm -1 exhibit stretching vibration of carbonyl group (C=O) in hard segments and C-O vibration corresponding to alcohol groups were observed at 1220 cm -1 , 1142 cm -1 , 770cm -1 (Ayyar et al., 2017).
Triacylglycerol composition (TAG) is the main compound of oils and fats, comprising 96-98% of them. The dominant TAGs in black seed oil are mainly tri-linoleoyl, oleoyl-di-linoleoyl, palmitoyldi-linoleoyl, palmitoyl-oleoyllinoleoyl and dioleoyl-linoleoyl (Mazaheri et al., 2019). It was supported by the fatty acid composition of BSO results (Table 4). It is well known that TAGs response to characteristic peaks were at 3009 cm -1 (trans =C-H stretch), 2923 cm -1 (-CH 2 symmetrical stretching), 2854cm -1 (-CH 2 asymmetrical stretching), 1743 cm -1 (-C=O stretching of esters) and 1659 (-C=C asymmetrical stretching) due to the dominance of carbon chains in the unsaturated fatty acids (Nurrulhidayah et al. 2011; Kalhori et al., 2018;Yang et al., 2005). The PU/BSO nanofiber spectrum was similar to PU nanofiber spectrum. The peak at 3325 cm -1 and 2951 cm -1 related to stretching vibration of the N-H and C=N which are characteristic bond of PU. But, the peak at 2926 cm -1 and 2855 cm -1 were due to the presence of oil in the nanofiber.

CONCLUSION
BSO is widely used for medical applications due to its anti-inflammatory, and antimicrobial properties. PU is a biocompatible, non-toxic, and non-inflammatory polymer that has been intensively used in wound dressings. Polymeric nanofibers provide excellent surfaces for wound healing because of their unique properties.
In the study, PU and PU/BSO nanofibrous mats were produced by electrospinning. The effect of BSO content, and electrospinning parameters such as flow rate and spinning distance on the morphological properties of nanofibrous mats were investigated.
SEM results showed that pure PU nanofibers had diameters between 400-700 nm. With the increasing flow rate, diameters of the PU nanofibers were increased, whereas the diameters were decreased with the increasing spinning distance.
Depending on the BSO content in the polymer solution, the nanofiber diameters also increased due to the increase in viscosity. The lowest diameter was obtained with the samples produced from 5% wt. BSO containing solutions.
BSO addition also had an effect on the wettability of the nanofibrous mats. Although BSO has a hydrophobic nature, contact angle values were decreased with the increase in BSO content due to the surface morphology. This indicates that not only the chemical structure of the BSO, but also the porosity play an important role on wettability of the nanofibrous mats. FTIR results showed that, BSO was successfully loaded into the nanofibrous structure and it was compatible with PU.
It can be concluded that, PU/BSO nanofibrous mats can be promising for wound healing applications due to their high porosity, reasonable wetting property, and small fiber diameters.