Fabrication of a Polycaprolactone/Chitosan Nanofibrous Scaffold Loaded with Nigella sativa Extract for Biomedical Applications

In this study, biocompatible electrospun nanofiber scaffolds were produced using poly(-caprolactone (PCL)/chitosan (CS) and Nigella sativa (NS) seed extract, and their potential for biomedical applications was investigated. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), total porosity measurements, and water contact angle measurements were used to evaluate the electrospun nanofibrous mats. Additionally, the antibacterial activities of Escherichia coli and Staphylococcus aureus were investigated, as well as cell cytotoxicity and antioxidant activity, using MTT and DPPH assays, respectively. The obtained PCL/CS/NS nanofiber mat was observed by SEM to have a homogeneous and bead-free morphology, with average diameters of 81.19 ± 4.38 nm. Contact angle measurements showed that the wettability of the electrospun PCL/Cs fiber mats decreased with the incorporation of NS when compared to the PCL/CS nanofiber mats. Efficient antibacterial activity against S. aureus and E. coli was displayed, and an in vitro cytotoxic assay demonstrated that the normal murine fibroblast cell line (L929 cells) remained viable after 24, 48, and 72 h following direct contact with the produced electrospun fiber mats. The results suggest that the PCL/CS/NS hydrophilic structure and the densely interconnected porous design are biocompatible materials, with the potential to treat and prevent microbial wound infections.


Introduction
Electrospinning is an efficient and practical method that utilizes a powerful electric force to move polymeric solutions to create micro-and nanofibers. Surface tension is deformed by the high voltage of an electrically conductive fluid in the fabrication of fibers, which have recently been investigated for their potential in biomedicine, such as in wound treatment and tissue engineering [1][2][3][4][5]. In practical electrospinning, a variety of variables, including melt or solution quality, electrospinning device parameters, and environmental variables, might influence the shape and characteristics of the resulting fibers [6,7]. However, to understand the effects of such complicated parameters on nanofibers when using SEM (MIRA TESCAN, Czech Republic) was used to examine the morphology of the composite nanofibrous scaffold at a 15 kV accelerating voltage. The scaffolds were coated with gold before imaging using a sputter coater with a 15 kV acceleration voltage and a magnification scale of 100,000× g. Scaffold fiber diameters were calculated using image analysis software based on SEM images at 5000× g magnification (ImageJ, U.S. National Institutes of Health, Bethesda, Maryland, USA).

Infrared Spectroscopy with Fourier Transform
PCL/PLA/NS nanofibers were obtained by combining 1 mg of sample with 100 mg of KBr of NS and using FTIR spectroscopy to analyze the structure and chemical makeup of the nanofiber, as well as any potential interactions between the extract and polymer in the nanofiber formations. In the 500-4000 cm −1 region, sample spectra were captured.

Water Contact Angle and Mechanical Properties
The water contact angle and mechanical features of the scaffolds were assessed. The contact angle was dynamically calculated using the Wilhelm plate approach [38]. The hydrophilicity or hydrophobicity of the sample was tested and evaluated using water with a surface tension of 72 dyn/cm. The liquid in the container increased until the metal plate's intended surface was completely submerged in water, at which point it began to descend. We determined the porosity of the fibers by weighing the samples and soaking them in PBS for 24 h. The weight was once more measured after the surface water and nanofiber web were eliminated from the solution. The nanofibrous scaffold's porosity was determined by the volume of liquid that it could retain.

In Vitro Cell Culture Studies
The MTT test was carried out to identify the cytoprotective criteria of PCL/CS/NS nanofibrous mats under conditions of oxidative stress. To encourage cell adhesion on the nanofiber surface, 1 cm 2 nanofibrous mats were sterilized overnight in a laminar flow hood under UV light for both the top and bottom surfaces. After being rinsed with distilled water and PBS to remove any remaining solvent, they were then submerged in DMEM overnight. Cells from L929 were cultured in DMEM supplemented with 10% FBS and 1% antibiotics at 37 • C and 5% carbon dioxide. The L929 cells were trypsinized and seeded at 1 × 10 5 cells per well onto the nanofibrous mat once they had attained % confluence. They were then incubated at 37 • C and 5% CO 2 . The MTT assay was used to test cell viability on the nanofibrous mats for 24, 48, and 72 h time periods.

Antibacterial Activity
The Kirby-Bauer disk diffusion method to test antibacterial activity (Humphries et al., 2018) [39] was considered a suitable method for evaluating the antibacterial activity of PCL/CS/NS nanofibers. Briefly, five bacterial colonies of S. aureus (ATCC 29213) and E. coli (ATCC 35218) were used to collect sterile inoculating loops, which were then suspended in 2 mL of sterilized PBS. By diluting the bacterial suspension with sterile PBS, the turbidity was already reduced to a 0.5 McFarland level. Inoculum channels were populated with sterile swabs. Bacterial swabs were inoculated into plates of Muller-Hinton agar. To disperse the nanofibers, we dissolved 0.1 mg PCL/CS/NS nanofibers in 1 mL of distilled water. Before use, the suspension was sonicated for 10 min. The standard was impregnated with 35 µL of the PCL/CS/NS nanofiber suspension, chitosan suspension, Nigella sativa extract (NS), distilled water (as a negative control), and antibiotic (as a positive control).

Activity of DPPH Radical Scavenging
This test was performed according to Blois's description (1958) [40]. A total of 0.025 g/L of DPPH was dissolved in methanol. Dimethyl sulfoxide (DMSO) was used to dilute various concentrations of chitosan (CS), Nigella sativa (NS), nanofibers (PCL/CS/NS), and ascorbic acid (as a control) to create a sample solution. Following the addition of 5 µL of the sample solution to each well of a 96-well plate, 195 µL of the DPPH working suspension was pipetted. The reaction took place at room temperature for 20 min, and the solution's absorbance at 515 nm was determined. The free radical scavenging activity of each fraction was assessed by comparing its absorbance to that of a blank solution (no sample). The following equation was used to calculate the percentage of inhibition, representing the capacity to scavenge DPPH radicals.

DPP Scavenging Activity
A 0 refers to the absorbance value of the control and A 1 represents the absorbance value of the test sample.

Morphology of Nanofibers
SEM micrographs showed the prepared PCL/CS/NS at various weight ratios. From Figure 2, the nanofiber mat had a more uniform and thinner texture, demonstrating that a uniformly favorable solution viscosity was achieved during electrospinning by using the optimized operating parameters. The current study results revealed nanofibers of a diameter between 40 ± 2.03 and 180 ± 2.16 nm, with an average of 101.85 ± 4.38 nm. The morphology of the prepared electrospun nanofibers was directly influenced by parameters such as the voltage, flow rate, and the distance from the tip to the collector.

FTIR Analysis
FTIR analysis was used to determine how electrospinning altered the parts that made up the PCL/CS/NS. The spectra of the PCL/CS/NS are shown in Figure 3. Prior research [57] revealed that the asymmetric and symmetric CH2 stretching peaks of the pure PCL membrane were at 2955 cm −1 and 2875 cm −1 , the CO stretching peak was at 1735 cm −1 , the asymmetric COC stretching peak was at 1260 cm -1 , and the symmetric CH2 stretching peak was at 1164 cm −1 (CC stretching). The broad peak at 3451 cm −1 that distinguished PCL from the PCL/CS/NS spectrum was caused by the stretching vibration of -OH and -NH2 from CS [58]. However, the PCL/CS/NS composite membrane's peaks were increased to 1500 cm −1 as a result of the peaks in the visible region of Figure 4 that belonged to NS [59]. This demonstrated that bioactive materials were electrospun into the PCL/CS/NS composite. CS and NS have many polar groups, such as -NH2 and -COOH, which have the potential to carry positive or negative charges and create a polyanion-polycation complex. A larger charge density on the surface would result in more elongation forces being applied to the released jet [46]. Additionally, the increased charge density may increase the jet bending instability, resulting in a reduced fiber diameter [47]. Incorporating N. sativa into PCL/CS nanofibers resulted in shifting the fibers' diameter and diameter distribution to lower values, which was in agreement with other previous reports [33].
The mechanical properties of PCL nanofibers have been enhanced through the addition of various fillers, including nanosilicates, graphene, cellulose nanocrystals, and Ag nanoparticles [48,49]. The mechanical strength of PCL nanofibers for biomedical applications has also been improved by blending them with natural or synthetic polymers [50]. Wang et al. (2021) [51] reported that adding natural polymers such as chitosan, alginate, and lignin to PCL nanofibers can improve their structural integrity, and hence their functionality. Since thymol was the active component of the NS extract, it stands to reason that this extract could serve as a plasticizer and regulator of the polymer chains, resulting in a reduction in the diameter of the nanofibers [52].
The shear viscosity of the spinning solution is often believed to be the key variable of the fiber diameter [53]. When the viscosity is too low, polymeric fibers and droplets of the material (electrospray) may be interrupted, while, when the viscosity is too high, the polymeric material cannot be extruded [54]. The needed minimum viscosity threshold varies with the molecular weight of the polymer and the type of solvent being employed and correlates with a certain polymer concentration in the electrospun solution [55]. PCL and CS are polymers with significantly different chemical properties and finding a common solvent to create a film was an important challenge. Additionally, it was crucial to maintain the optimal viscosity in order to create the double porous membrane structure. By increasing the chitosan ratio, it was possible to electrospin PCL/chitosan blends, and SEM pictures revealed that as the chitosan ratio increased, the fiber diameter and dispersion reduced. According to Roozbahani, Fatemeh et al., PCL-treated chitosan nanofibers with a 70/30 ratio have a smaller average diameter of 205 nm than blended nanofibers made from untreated chitosan, which has a 356 nm diameter [56].

FTIR Analysis
FTIR analysis was used to determine how electrospinning altered the parts that made up the PCL/CS/NS. The spectra of the PCL/CS/NS are shown in Figure 3. Prior research [57] revealed that the asymmetric and symmetric CH2 stretching peaks of the pure PCL membrane were at 2955 cm −1 and 2875 cm −1 , the CO stretching peak was at 1735 cm −1 , the asymmetric COC stretching peak was at 1260 cm −1 , and the symmetric CH2 stretching peak was at 1164 cm −1 (CC stretching). The broad peak at 3451 cm −1 that distinguished PCL from the PCL/CS/NS spectrum was caused by the stretching vibration of -OH and -NH2 from CS [58]. However, the PCL/CS/NS composite membrane's peaks were increased to 1500 cm −1 as a result of the peaks in the visible region of Figure 4 that belonged to NS [59]. This demonstrated that bioactive materials were electrospun into the PCL/CS/NS composite.

Water Contact Angle and Porosity Results
Wettability is an essential factor to consider when choosing a wound dressing since it influences cell adherence, proliferation, and the ability to absorb exudates. The water contact angle can be used to determine the wettability of a surface. The water contact angle was measured to determine the behavior of the composite PCL/CS/NS mats and to assess keratinocytes and fibroblasts, at concentrations of 1-1000 μg/mL [61]. Given these findings, it is reasonable to conclude that the PCL/CS/NS scaffold, at the ratio of 3/1/2, is a good option for cell culture because it increased the rate of proliferation of L929 cells over time [62]. This is consistent with the findings of Uddin et al. 2022 [63], who proved that, according to their MTT results, NS-containing composite mats were non-cytotoxic and increased fibroblast migration and proliferation.

Antibacterial Activity
An antibacterial evaluation was conducted using a disk diffusion technique for each bacterium. The diameter of the inhibition zone was measured after 24 h of incubation by a caliper. As shown in Figure 6, the prepared PCL/CS nanofiber mats containing NS had better antibacterial properties than PCL and PCL/CS. The results of the current study showed a notable inhibition zone of 8.00 mm ± 0.22 and 7.4 mm ± 0.16 for S. aureus and E. coli, respectively. The inhibition zone diameter was used as an index of the scaffold's an-

Water Contact Angle and Porosity Results
Wettability is an essential factor to consider when choosing a wound dressing since it influences cell adherence, proliferation, and the ability to absorb exudates. The water contact angle can be used to determine the wettability of a surface. The water contact angle was measured to determine the behavior of the composite PCL/CS/NS mats and to assess the hydrophilicity alterations in the nanocomposite scaffolds. As presented in Table 2, the PCL film exhibited poor hydrophilicity, with an average contact angle of 122.5 • , which was in line with the hydrophobic nature of the polymer. The contact angle value of PCL at 8% decreased to 99.4 • , and then to 53.2 • , after the addition of CS at 2% and NS at 10%, respectively. Results of the wettability test demonstrated that the incorporation of NS and CS within the PCL matrix may have produced some hydrophilic groups, such as NH and OH, on the surfaces of the nanocomposite membranes. The results of the mechanical properties are displayed in Table 3. The PCL/CS/NS nanofiber mat's tensile strength was 5.4 ± 0.2 MPa, which was higher than the range of 1.8 ± 0.1 MPa for the PCL nanofibers alone, and was in line with earlier studies [60].

In Vitro Cell Culture Studies
The MTT test was used to determine the impact of PCL/CS/NS nanofiber scaffolds on the viability of L929 cells. The viability of the PCL/CS/NS scaffolds is shown in Figure 5 at 24, 48, and 72 h. As shown in the graph, the growth rate of the PCL/CS/NS nanocomposite scaffold was significantly higher than that of PCL, PCL/CS, and PCL/NS, and it approached that of the control sample by the end of the third day. The PCL/CS/NS scaffold's fibers had very small diameters compared to pure PCL fibers, providing an appropriate space for cells to be placed. Furthermore, according to the test for determining scaffold hydrophilicity, adding NS to the polymer solution significantly increased the scaffold's hydrophilicity, resulting in better cell adhesion to the scaffold. In a study conducted by Zagórska-Dziok, Martyna et al., N. sativa was found to have no cytotoxic effect on keratinocytes and fibroblasts, at concentrations of 1-1000 µg/mL [61]. Given these findings, it is reasonable to conclude that the PCL/CS/NS scaffold, at the ratio of 3/1/2, is a good option for cell culture because it increased the rate of proliferation of L929 cells over time [62]. This is consistent with the findings of Uddin et al. 2022 [63], who proved that, according to their MTT results, NS-containing composite mats were non-cytotoxic and increased fibroblast migration and proliferation. tibacterial activity in the disk diffusion test; the inhibition zone diameter of the mats containing PCL/CS/NS against S. aureus (Gram-positive) was greater than for E. coli (Gramnegative bacteria). Ciprofloxacin at 10 μg/mL was used as a positive control. According to the antibacterial activity test results of the present study, the inclusion of NS in the composite scaffold promoted antibacterial activity. Gram-positive bacteria are sensitive to these mats, and these results were in agreement with a result previously reported by Shahverdi et al. 2022 [64]. The antibacterial action of N. sativa seed extract may cause bacterial cell membranes to become permeable, resulting in cell destabilization and death [65]. Gram-negative bacteria are more resistant because their cell membranes are double-layered, as opposed to Gram-positive bacteria's single-layer membranes [66,67].  Figure 7 depicts the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method used to assess the antioxidant properties of the prepared nanofibrous mats. The free radical scavenging capacities were measured using the DPPH assay. Scavenging is most effective with electron or hydrogen donor scaffolds that quench and stabilize DPPH to DPPH-H. NS-containing scaffolds demonstrated dose-dependent scavenging potency comparable to ascorbic acid (p ≤ 0.005) ( Table 4). Large amounts of ROS are produced during inflammation, causing biological damage such as lipid, protein, and nucleic acid degradation, and ultimately cell death, which disrupts the recovery process. The use of antioxidants can significantly aid enzymatic repair and metabolism [68]. Many studies have demonstrated that biogenic nanomaterials are a consistent source of antioxidant activity [69,70].

Antioxidant Activity
Previous studies have experimented with various extraction methods, including the DPPH scavenging assay, for extracts of N. sativa seeds [71]. In addition to beta-sitosterol, the NS seed extract contains significant amounts of other antioxidants, such as various tocopherol and tocotrienol isomers found in the alpha, beta, gamma, and delta forms. Another study found that an ethanolic extract of N. sativa seeds inhibited the DPPH scavenging assay by a higher percentage than a methanolic extract, which inhibited the assay by only 3.77% [72].
The current study's findings are in excellent agreement with those of other investigations. Arif et al. (2021) [73] found that nanosuspensions of N. sativa extracts had the

Antibacterial Activity
An antibacterial evaluation was conducted using a disk diffusion technique for each bacterium. The diameter of the inhibition zone was measured after 24 h of incubation by a caliper. As shown in Figure 6, the prepared PCL/CS nanofiber mats containing NS had better antibacterial properties than PCL and PCL/CS. The results of the current study showed a notable inhibition zone of 8.00 ± 0.22 mm and 7.4 ± 0.16 mm for S. aureus and E. coli, respectively. The inhibition zone diameter was used as an index of the scaffold's antibacterial activity in the disk diffusion test; the inhibition zone diameter of the mats containing PCL/CS/NS against S. aureus (Gram-positive) was greater than for E. coli (Gram-negative bacteria). Ciprofloxacin at 10 µg/mL was used as a positive control. According to the antibacterial activity test results of the present study, the inclusion of NS in the composite scaffold promoted antibacterial activity. Gram-positive bacteria are sensitive to these mats, and these results were in agreement with a result previously reported by Shahverdi et al. 2022 [64]. The antibacterial action of N. sativa seed extract may cause bacterial cell membranes to become permeable, resulting in cell destabilization and death [65]. Gram-negative bacteria are more resistant because their cell membranes are double-layered, as opposed to Gram-positive bacteria's single-layer membranes [66,67].
BioTech 2023, 4, x FOR PEER REVIEW 11 of 15 radical scavenging activity was greatly enhanced by increasing the quantities of the nanosuspensions and N. sativa extracts. The maximal amount of free radical scavenging activity for nanosuspensions of N. sativa extract was seen at 500 g/mL, according Ali et al. [74]. Thus, the DPPH free radical scavenging activity was dramatically improved by increasing the quantities of the nanofibrous mat.    Figure 6 depicts the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method used to assess the antioxidant properties of the prepared nanofibrous mats. The free radical scavenging capacities were measured using the DPPH assay. Scavenging is most effective with electron or hydrogen donor scaffolds that quench and stabilize DPPH to DPPH-H. NS-containing scaffolds demonstrated dose-dependent scavenging potency comparable to ascorbic acid (p ≤ 0.005) ( Table 4). Large amounts of ROS are produced during inflammation, causing biological damage such as lipid, protein, and nucleic acid degradation, and ultimately cell death, which disrupts the recovery process. The use of antioxidants can significantly aid enzymatic repair and metabolism [68]. Many studies have demonstrated that biogenic nanomaterials are a consistent source of antioxidant activity [69,70].

Antioxidant Activity
Previous studies have experimented with various extraction methods, including the DPPH scavenging assay, for extracts of N. sativa seeds [71]. In addition to beta-sitosterol, the NS seed extract contains significant amounts of other antioxidants, such as various tocopherol and tocotrienol isomers found in the alpha, beta, gamma, and delta forms. Another study found that an ethanolic extract of N. sativa seeds inhibited the DPPH scavenging assay by a higher percentage than a methanolic extract, which inhibited the assay by only 3.77% [72].
The current study's findings are in excellent agreement with those of other investigations. Arif et al. (2021) [73] found that nanosuspensions of N. sativa extracts had the highest free radical scavenging activity of up to 55% at doses of 1000 mg/mL, and the lowest activity of up to 28% at 250 mg/mL. This work demonstrated that the DPPH free radical scavenging activity was greatly enhanced by increasing the quantities of the nanosuspensions and N. sativa extracts. The maximal amount of free radical scavenging activity for nanosuspensions of N. sativa extract was seen at 500 g/mL, according Ali et al. [74]. Thus, the DPPH free radical scavenging activity was dramatically improved by increasing the quantities of the nanofibrous mat.

Conclusions
According to the research's findings, Nigella sativa-loaded PCL/CH electrospun nanofibers formed a new nanofibrous scaffold that was discovered to be non-toxic to skin L929 fibroblast cells. The production method resulted in thin fibers with mean diameters as low as 82 nm, high porosity, promising tensile strength, enhanced hydrophilicity, and biocompatibility. The incorporation of N. sativa into the PCL/CH matrix was supported by the findings of the chemical investigation of the nanofibrous composite by FTIR spectroscopy and structural XRD analysis.
The results of the cell viability test, which showed this formulation's great biocompatibility, were supported by the MTT assay. The inclusion of Nigella sativa extract also decreases the diameter of nanofibers. It also improves the antioxidant and antibacterial properties.  Data Availability Statement: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.