Int J App Pharm, Vol 16, Issue 5, 2024, 99-110Original Article
DEVELOPMENT AND OPTIMIZATION OF THE WOUND HEALING ELECTROSPUN POLYURETHANE/COLLAGEN/PHYTOCERAMIDES NANOFIBERS USING THE BOX-BEHNKEN EXPERIMENTAL DESIGN (QUALITY BY DESIGN)
TASSNEIM M. EWEDAH1*, MOHAMED EL-NABARAWI2*, MAHMOUD H. TEAIMA2, SAMMAR FATHY ELHABAL3, KAMEL R. SHOUEIR4,5, ABDALLAH M. HAMDY6, AHMED ABDALLA1
1Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Egyptian Russian University, Badr City, PO, Cairo-11829, Egypt. 2Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, PO, Cairo-11562, Egyp. 3Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Modern University for Technology and Information (MTI), Mokattam, Cairo-11571, Egypt. 4ICPEES, Institut de Chimie et Procédé pour l’Energie, l’Environnement et la Santé, CNRS, UMR 7515, Université de Strasbourg, 25 Rue Becquerel-67087 Strasbourg Cedex 2, France. 5Institute of Nanoscience and Nanotechnology, Kafrelsheikh University, Kafrelsheikh-33516. 6Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, PO, Cairo-11829, Egypt
*Corresponding author: Tassneim M. Ewedah; *Email: tassneimewedah@gmail.com
Received: 20 May 2024, Revised and Accepted: 03 Jul 2024
ABSTRACT
Objective: This study aimed to develop and optimize polyurethane/collagen/phytoceramides nanofibers, a wound-healing drug delivery approach, using the electrospun technique. The objective was to enhance the effectiveness of nanofibers by optimizing the preparation process.
Methods: The box-behnken design was established to optimize the electrospinning instrument performance and, consequently, the nanofiber effectiveness. Response variables were diameter, zeta potential, and diffusion coefficient, while the experimental key factors were applied voltage, flow injection rate, and rotary collector speed of the electrospinning instrument. The optimized nanofibers were examined to ensure the validity of the optimization process.
Results: The study built prediction models for each response and employed a desirability function to suggest an optimum working level of each factor that guarantees minimum diameter, maximum zeta potential, and maximum diffusion coefficient. The desirability function suggested experimental conditions of 12.9 KV for the applied voltage, 1.3 ml/h for the injection flow rate, and a speed of 920 rpm for the rotary collector speed. The optimized formula proved satisfactory physicochemical properties regarding the nanofiber's infrared spectrum and wettability characteristics. The biomedical effectiveness of the optimized nanofibers showed increased anti-inflammatory potency up to 82.8±2.6% and a high wound closure rate of about 79%. Also, the stability study showed a nonsignificant change in response over the studied points.
Conclusion: The optimized nanofiber formula achieved the desired diameter, zeta potential, and diffusion coefficient. The results proved the Box-Behnken design approach's efficacy in enhancing the nanofiber formula's effectiveness and stability.
Keywords: Nanofiber, Wound healing, Box behnken design, Optimization, Quality by design
© 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2024v16i5.51510 Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
Inflammation, proliferation, and remodelling are all steps in the intricate biological process of wound healing. Being the biggest organ in the body, the skin is vital in this process because it acts as a natural defence mechanism against harmful substances [1, 2]. A multi-step and dynamic process is initiated at the site of a skin injury, whether acute or chronic, when the skin's integrity is compromised. Repairing wounds entails balancing inflammatory cytokines at the site of the injury and restoring tissue integrity [3].
A unique combination of polyurethane, collagen, and phytoceramides for tissue engineering purposes was prepared. Electrospun nanofibers of polyurethane, collagen, and phytoceramides improved cell adhesion and elongation adaptability adaptability. A revolutionary idea for tissue engineering is introduced by combining these three polymers. The hydrophobic character of polyurethane is responsible for wound healing since it raises surface tension and, hence, lessens the contact between cells and the scaffold. Because they cannot connect with nearby cells, stressed cells take on a spherical shape and are unable to expand themselves like fibroblasts or carry out regular growth and reproduction [4]. Collagen is leading the way in wound treatment because it meets all of these criteria: it is ubiquitous, it has low immunogenicity, and it can be bent into strong, biocompatible scaffolds.
Additionally, collagen-based materials expertly blend synthetic and natural macromolecules. Collagen is essential for providing tissues with both mechanical strength and flexibility. Furthermore, it is a favourable surface for cellular attachment, growth, and specialization. Collagen is used as a surface coating to improve moisture preservation and facilitate cell attachment in scaffold matrices [5-14]. Ceramides are influential in facilitating cellular effects by activating cell surface receptors, promoting cell proliferation and migration, and strengthening the skin's protective barrier [15–17].
Quality by design is an approach used in various studies to ensure an ongoing supply of high-quality products. Design of Experiments (DOE) introduces data-based evidence leading to comprehension processes from the least number of experiments [18–20]. The nanofiber preparation procedure has a critical effect on the shape and morphology of the nanofiber and, consequently, its effectiveness. The preparation process includes several parameter factors that must be controlled to obtain more effective and stable nanofibers. Several works represented the effect of different factors on the response variables of the prepared nanofibers [21–27]. However, no previous work discussed the optimization process via compromising between all factors to obtain the most optimum conditions in the preparation process, thus ensuring the best effectiveness and stability.
The current work discusses the optimization of the nanofiber preparation process. The work involves establishing a Box-Behnken design to predict the optimum experimental conditions for preparing nanofibers. The prepared optimized nanofibers were examined in terms of physicochemical properties and biomedical effectiveness. A stability study was also performed on the dosage form before and after optimization.
MATERIALS AND METHODS
Materials
The thermoplastic polyurethane (1185A), with a density of 1.12 g/cm3, is supplied by BASF Elastollan in Germany. Vita Nuova, a company based in the United Kingdom, provides support for phytoceramides (Lipowheat, 350 mg). Triton X-100 and N, N-dimethylformamide (DMF), which is anhydrous and has a purity of 99.8%, with a chemical formula of C3H7NO, a solubility parameter (δ) of 12.14 (cal/cm3).5, and a dielectric constant (ε) of 37, were obtained from Sigma. Sodium hydrogen phosphate (Na2HPO4), glacial acetic acid (CH3COOH, 99-100%), and ethylene-diaminetetraacetic acid (EDTA, (HO2CCH2)2NCH2CH2N(CH2CO2H)2) were obtained from MERCK.
Extraction of collagen
Collagen was extracted from a fresh piece of tilapia fish (Oreochromis sp.). The tendons were dissected into small fragments following thorough cleaning. The tendon was treated using a 0.05 M aqueous solution of Na2HPO4 at a temperature of 5 °C for two days. Afterwards, the tendon underwent a washing procedure using Phosphate-Buffered Saline (PBS). Next, the tendon was subjected to a solution consisting of 0.5 M acetic acid, pepsin at a concentration of 1.0 g/100 g of tissue, and a 3 mmol EDTA solution. The procedure was conducted at a temperature of 5 °C for 6 d. The diluted solution underwent centrifugation at a speed of 6000 revolutions/min for 15 min. Combine a 4 M aqueous solution of NaCl with the supernatant while stirring. Following filtration, the collagen gel solution was subjected to dialysis using distilled water at a temperature of 5 °C for two weeks until it reached a pH of 7. The freeze-drying process was carried out by subjecting the samples to a temperature of-60 °C for 24 h.
Procedures
Electrospinning of polyurethane/Collagen/Phytoceramides nanofibers (Preliminary formula)
Fabrication of polyurethane/Collagen/phytoceramides nanofibers
Before use, the polyurethane granules were subjected to a drying process in a vacuum oven at a temperature of 80 °C for 1 h to remove any moisture that had been absorbed. Afterwards, a solution was created by dissolving 3.5 g of polyurethane granules with a weight concentration of 7 weight percent in 50 milliliters of DMF solvent. As a way of fully dissolving and creating a uniform solution without any solid additives, the mixture was agitated using a magnetic stirrer for 3 h at normal room temperature. The polyurethane that had been dissolved was subsequently mixed with extracted collagen at a concentration of 5 w % concerning the polyurethane. Each mixture was agitated for an additional 4 h at room temperature until a uniform dispersion was achieved, subsequently, in the production of an electrospun polyurethane/collagen/ phytoceramides fibrous film, a phytoceramides quantity of 1.5 wt. % of the solution's total weight was added to the prepared polyurethane/collagen mixture. This mixture was then transferred to the electrospinning unit. The electrospinning process was carried out with parameters including a voltage of 18 KV, a flow rate of 1.7 ml/h, a rotary speed of 650 rpm, and a distance between the needle tip and the collector of 14 cm. Subsequently, after spinning 10 ml of polyurethane/collagen/ phytoceramides dispersion, the nanofibers were carefully deposited on an aluminium-coated collector. The resulting fibers were dehumidified in a vacuum desiccator and carefully stored for later analysis.
Measurements of the prepared Polyurethane/Collagen/ Phytoceramides nanofibers
Response variables, namely, diameter, zeta potential, and diffusion coefficient, were determined for the prepared nanofibres.
The functionalities of nanofibers in cell adhesion, proliferation, and tissue regeneration were shown to be significantly influenced by their diameter. Like the extracellular matrix, smaller diameters improve cellular interactions, tissue integration, and regeneration in biomedical scaffolds. The optimization of their performance for particular applications in tissue engineering, drug delivery, and wound healing still heavily depends on precisely controlling and manipulating their diameter [28]. Thus, the morphology of the nanofibrous scaffolds and the fiber diameter were analyzed via Scanning Electron Microscopy (SEM) using ImageJ (NIH Image) software 1.8.0, Maryland, USA.
Electrostatic repulsion occurs between nanofibers possessing a high zeta potential. This repulsion increases the wetting and spreading of the liquid on the fiber surface. As a result, higher zeta potential fibers typically show better liquid absorption and increased wettability [29].
The zeta potential and translational diffusion coefficient measurements of dispersions containing nanofibers were carried out using a dynamic light scattering instrument (Malvern Zetasizer Nano ZS, Worcestershire, UK) at 25 ○C in duplicate. All samples were dispersed in ethanol at a concentration of 0.1% (w/v) for both measurements.
Optimization process
Box behnken design
Box Behnken Design (BBD) was established to optimize nanofibers. The Box-Behnken design was developed using Stat graphics program version 16, where levels were carefully selected (3 levels for each factor). A set of 15 nanofiber preparations was performed according to the multilevel multifactor Box-Behnken design. The chosen design consists of fifteen runs [30], with a single sample being taken for each run. The default model is quadratic with 10 coefficients. The optimal setting of the experimental factors has been determined and is displayed.
Prediction and preparation of optimized nanofiber formula
Following the same procedures as the preliminary formula, the intended responses of nanofibers were measured for the preparations fabricated according to BBD to obtain the results used in predicting the optimum nanofiber conditions using Stat Graphics program version 16.
An estimated response surface was constructed for each response variable to obtain the optimum values statistically.
The desirability function was utilized to collectively forecast the optimal values for all response variables. Subsequently, the optimal values for all factors were concluded, and the optimized nanofiber was prepared.
Verification of the optimized nanofiber formula
The optimized nanofiber was assessed by measuring different responses in the same manner to compare the results with those of the predicted response variables (predicted by design). Calculations were made to compare the actual results with the predictions.
Evaluation of the prepared nanofiber formulas
Fourier transform infrared spectroscopy (FTIR)
Fourier transform infrared spectroscopy (FTIR) IR Affinity-1SFTIR Spectrophotometer, Japan, was used to investigate the chemical composition and newly created linkages between the electrospun components. Samples are scanned at 4 mm/s at a resolution of 2 cm over a wave number region of 4000–400 cm-1. The obtained Infrared (IR) data are interpreted to examine nanofiber properties [31].
Wetting properties
The precise range of hydrophilicity in the constructed scaffolds is essential for promoting cells' initial attachment and movement. The water-attracting capabilities of the scaffolds were evaluated by measuring the static contact angle. The surface contact angle of electrospun nanofiber scaffolds was determined using the drop-shape analysis system Theta Optical Potentiometer (T-200 Biolin Sci., Finland). Exactly 200 microliters of clean water were delicately placed onto the nanofiber surface and meticulously adjusted to align with the camera's viewpoint. Five individual drops were precisely placed in specific areas on nanofibrous scaffolds measuring 1.5 × 1.5 cm2 for each sample [32].
Cell viability
The vitality of human skin fibroblast (HSF) cells, supplied by Nawah Scientific Research Center, Cairo, Egypt, was assessed by detecting the color change of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) from yellow to purple formazan. The efficiency of this conversion process relies on the optimal performance of mitochondria. The experimental methods were carried out in a sterile setting. The cells were cultured in several successive batches for 10 d. Subsequently, the cells were introduced into the wells of 96-well plastic plates at a density of 10x103 cells per well, using a fresh complete growth medium. The plates were placed in a 5% CO2 incubator, which was maintained at a temperature of 37 °C for 24 h. Two sets of wells were created for the experiment. The first group served as the negative control and contained only cells. The second group comprised cells subjected to varying concentrations (2500, 1250, 625, and 312.5 ng/ml) of possible nanofiber compositions.
Following an additional incubation period of 48 h, the liquid medium was extracted from each well. Subsequently, a volume of 20 microliters of MTT salt solution at a concentration of 2.5 μg/ml was introduced into each well. Subsequently, the plate was placed in an incubator and kept at a temperature of 37 °C in an environment enriched with 5% CO2 for an extra 4 h duration. We added 200 μl of a solution containing 10% sodium dodecyl sulfate (SDS) in 0.01M HCL to each well to stop the reaction and scatter the generated crystals. The plates were thereafter placed in a 5% CO2 incubator and incubated overnight at a temperature of 37 °C. Positive control was employed to guarantee total cell death under the same conditions. The control utilized a cytotoxic natural material with a concentration of 100 µg/ml. The measurement was conducted at a wavelength of 595 nm, using a reference wavelength of 620 nm [33].
Antioxidant activity
When the compound 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) comes into contact with a material that removes unstable molecules, it experiences a chemical reaction that reduces its oxidation state, causing its color to change from purple to yellow. This shift happens due to a decrease in the efficacy of the antioxidants present in the material. To evaluate the antioxidant characteristics of the prospective nanofiber formulations, a nanofiber sheet measuring 1x1 cm (100 mg) from each sample was submerged in a solution containing 10 ml of 0.1 mmol DPPH in methanol.
The specimens were then placed in a lightless incubator at a temperature of 37 °C for several periods, including 4, 8, and 12 h. The Ultraviolet-visible (UV-visible) spectrophotometer was utilized to measure the absorbance of the solutions at a wavelength of 515 nm at predetermined time intervals. The antioxidant potency was determined by applying the following equation:
….. [34]
In vitro anti-inflammatory potency
The anti-inflammatory efficacy of the nanofiber compositions was assessed in vitro using the protein denaturation method. This method was delineated in a prior publication with a few inconsequential modifications. Diclofenac sodium, a potent nonsteroidal anti-inflammatory medication, was employed as a reference standard. A 1x1 cm nanofibrous mat was introduced into a reaction mixture containing 2.0 ml of phosphate-buffered saline (pH 6.4) and 2 ml of albumin produced from freshly laid eggs (1 mmol). The solution was placed in an incubator and maintained at a temperature of 37 °C for 20 min.
The denaturation process was conducted by subjecting the solution to a temperature of 60 ºC in a water bath for 15 min. The absorbance was measured at a wavelength of 660 nm, with the experiment carried out at standard ambient temperature. The experiment was duplicated three times. The equation utilized to evaluate the inhibition of protein denaturation is as follows:
….. [35]
Wound healing assay
The migration of the cells was assessed using a wound scratch assay. We cultured the HSF cell line by placing 5 10 5 cells/well in a 6-well plate and allowing them to incubate overnight at 37 °C in an environment containing 5% CO2. Subsequently, the medium was extracted, and the adhering sheets of cells were gently scraped using a sterile 10 µl pipette tip, followed by rinsing with PBS. Subsequently, we introduced 3 milliliters of low serum medium with a concentration of 1% fetal bovine serum (FBS) in Dulbecco's Modified Eagle Medium (DMEM) as treatment. The cells were then subjected to drug treatment using dosages that corresponded to the IC50 values of each substance. Cell migration into the wound space was observed and recorded using an inverted microscope and optical camera (ZEISS ZEN microscope software, blue edition) at 0, 24, and 48 h after treatment. The wound closure rate was calculated [36].
Stability study
The responses were measured following the same procedures as the preliminary formula to assess the stability of the optimized nanofiber formula. This is considered a zero-point stability approach intended to be compared with the results of other months' stability study points using Analysis Of Variance (ANOVA) statistical analysis.
An accelerated stability study was implemented on the optimized and preliminary formulas before optimization. Studies on the 1 mo accelerated point, the 2 mo accelerated point, and the 3 mo accelerated point were performed and compared with the zero stability point. At every point of acceleration, each formula was exposed to stress conditions at a temperature of 40 ⁰C and Relative Humidity (RH) % = 75. The results were determined from different response variables (three experiments for each response) for each formula at each accelerated point. The study was also implemented at ambient temperature conditions at 1 mo, 2 mo, and 3 mo points.
A stability cabinet (40 °C and an RH of 75%) was used for the accelerated study purpose, where the prepared nanofibers were placed, and a sample was withdrawn every month for examination.
A statistical comparison using a two-way ANOVA within a 95% confidence interval [37] was performed on the results obtained from the stability study. Also, a one-way ANOVA analysis was performed to ensure the stability of the optimized formula. Statistical analysis was performed using SPSS® statistical software. Results were considered significant if the significance level was below 0.05.
RESULTS
A nanofiber drug delivery approach using polyurethane, collagen, and phytoceramides was prepared with the aim of wound healing. This combination is unique and has proven highly efficacious for tissue engineering purposes. Fig. 1 shows the main parts of the electrospinning instrument that is used for the preparation of nanofibers. The instrument parameters include a high-voltage power supply, an injection pump, and a rotary collector plate (collector drum).
This study aims to optimize the experimental conditions of the nanofiber preparation process. This involves computational steps to obtain the most effective and stable nanofiber formula.
The establishment of the Box Behnken design was performed in which levels of the selected factors, namely, voltage (X1), the flow injection rate (X2), and speed of the rotary collector drum (X3), were carefully selected (3 levels for each factor).
The optimized nanofiber was examined by measuring the diameter, zeta potential, and diffusion coefficient (Y1, Y2, and Y3, respectively). This study also involves the prediction, verification, and evaluation of the optimized nanofiber.
Nanofibers of different diameters and a linked fiber shape form randomly, as seen in fig. 2. The diameters of the nanofibers were measured by analyzing scanning electron micrographs with the ImageJ program.
Several fibers were chosen separately for each sample, and the diameters of each were recorded. The next step was determining the average diameter, which allowed for a thorough evaluation of the nanofiber parameters.
Fig. 1: Main parts of electrospinning instrument
Fig. 2: SEM photograph (a) and diameter distribution of nanofibers (b)
Optimization process
Box-behnken design
Factors used in the design were voltage (X1), a flow injection rate (X2), and the speed of the rotary collector (X3). The responses, namely diameter (Y1), zeta potential (Y2), and diffusion coefficient (Y3), were observed.
For each factor, three levels were selected carefully. For the applied voltage, the three levels were 20 KV for the high level, 16 KV for the medium level, and 12 KV for the low level.
The three levels for the injection flow rate factor were 1.8 ml/h for the high level, 1.5 ml/h for the medium level, and 1.2 ml/h for the low level.
For the speed of the rotary collector factor, the three levels were 1000 rpm for the high level, 600 rpm for the medium level, and 200 rpm for the low level.
The Box-Behnken design of the fifteen prepared formulas is expressed in table 1. The intended responses were measured (three measurements for each response variable) for all the Box-Behnken-prepared nanofibers. Results are represented in table 2.
Table 1: The different conditions for formulas prepared according to the box-behnken multilevel multifactor design
| Formula | Coefficient | Voltage | Flow rate | Drum collector speed |
| B1 | Axial point (middle) | 0 | 0 | 0 |
| B2 | B | -1 | 1 | 0 |
| B3 | CC (quadratic) | -1 | -1 | 0 |
| B4 | Axial point (middle) | 0 | 0 | 0 |
| B5 | AA (quadratic) | 0 | -1 | -1 |
| B6 | B | 0 | 1 | -1 |
| B7 | C | -1 | 0 | 1 |
| B8 | AC | 1 | 0 | 1 |
| B9 | A | 1 | -1 | 0 |
| B10 | C | 0 | -1 | 1 |
| B11 | BC | 0 | 1 | 1 |
| B12 | AB | 1 | 1 | 0 |
| B13 | A | 1 | 0 | -1 |
| B14 | Axial point (middle) | 0 | 0 | 0 |
| B15 | BB (quadratic) | -1 | 0 | -1 |
A is the voltage factor. B is the flow rate factor. C is the drum collector speed factor. AB, AC, and BC are the interaction terms between the factors. AA, BB, and CC are the quadratic terms of the factors. (1) High level. (0) Medium level. (-1) Low level.
Table 2: Variable response data from the fifteen formulas prepared according to the box-behnken multifactor design
| Formula | Diameter (nm) | Zeta potential (≈ mV) | Diffusion coefficient (µm2/S) |
| Mean±SD⁎ | |||
| B1 | 590±0.58 | 14±0.91 | 0.420±0.41 |
| B2 | 572±1.02 | 17±0.38 | 0.452±0.06 |
| B3 | 399±0.87 | 20±1.61 | 0.604±0.45 |
| B4 | 567±0.62 | 13±0.47 | 0.441±0.08 |
| B5 | 580±0.93 | 12±1.28 | 0.524±0.22 |
| B6 | 812±0.58 | 16±0.98 | 0.367±0.12 |
| B7 | 598±1.17 | 18±0.62 | 0.650±0.07 |
| B8 | 649±1.45 | 15±1.18 | 0.295±0.34 |
| B9 | 471±0.43 | 12±1.09 | 0.356±0.57 |
| B10 | 517±1.05 | 15±1.36 | 0.488±0.19 |
| B11 | 602±1.55 | 13±0.80 | 0.394±0.03 |
| B12 | 715±1.67 | 11±0.72 | 0.341±0.29 |
| B13 | 746±0.84 | 15±1.05 | 0.311±0.04 |
| B14 | 585±0.07 | 13±1.33 | 0.433±0.62 |
| B15 | 619±1.22 | 23±1.28 | 0.383±0.09 |
⁎Data are expressed as the mean±standard deviation of three experiments (n = 3).
Standard pareto charts
To represent the effect of different factors and their interactions (in descending order of significance) on the dependent variables, standard Pareto charts were constructed for all response variables, showing the significant factors that affect each response variable.
Statistical models have been fitted to the response variables. Models with P-values below 0.05 indicate that the model is statistically significant at the 5.0% significance level.
The results obtained by preparations designed by Box-Behnken design were statistically analyzed using ANOVA statistical analysis within a 95% confidence interval. The significant results observed for all response variables are recorded in table 3.
The ANOVA table partitions the variability in all response variables, namely, diameter, zeta potential, and diffusion coefficient, into separate pieces for each effect. It then tests the statistical significance of each effect by comparing the mean square against an estimate of the experimental error. In this case, effects have P-values less than 0.05, indicating that they are significantly different from zero at the 95.0% confidence level. As shown in table 3, the tabulated F values were found to be less than the calculated ones, indicating that a significant difference is present.
The values for the different response variables were analyzed, and a mathematical model for each response variable was constructed.
Effects on diameter
Effects on diameter (Y1) were studied. The Y1 response average ranged from 399 nm in B3 to 812 nm in B6, as shown in table 2. It was found that all factors are responsible for the difference in the diameter of the nanofiber, as displayed in the Pareto chart of Y1, fig. 3. Fig. 4 shows a direct relationship between both (X1) and (X2), and the diameter (Y1) while showing almost an inverse relationship between (X3) and the diameter (Y1). At the same level of X2 and X3 in B3 and B9, the rise in voltage from 12 KV to 20 KV was correlated with the increase in diameter average from 399 nm to 471 nm. Also, at the same level of X1 and X3 in B5 and B6, the rise in the flow injection rate from 1.2 ml/h to 1.8 ml/h was correlated with the increase in diameter average from 580 nm to 812 nm. In contrast, the rise of the speed of the rotary collector from 200 rpm in B5 to 1000 rpm in B10 was correlated with the decrease in diameter average from 580 nm to 517 nm at the same level of other factors X1 and X2.
Fig. 3: A standardized pareto chart of all response variables shows significant factors that affect each response variable in formulas prepared according to the box-behnken design
Table 3: ANOVA statistical analysis within a 95% confidence interval on response variables data obtained from the fifteen preparations designed according to the box-behnken design showing factors affecting each response
| Dependent variable: responses | ||||||
| Coefficient | Sum of squares | df⁎ | mean square | F⁎⁎ | p-value | |
| Diameter | Factor A: Voltage | 19306.1 | 1 | 19306.1 | 20.46 | 0.0063 |
| Factor B: Flow Rate | 67344.5 | 1 | 67344.5 | 71.39 | 0.0004 | |
| Factor C: Drum Collector Speed | 19110.1 | 1 | 19110.1 | 20.26 | 0.0064 | |
| CC | 23877.6 | 1 | 23877.6 | 25.31 | 0.0040 | |
| Total error | 4716.92 | 5 | 943.383 | |||
| Zeta Potential |
Factor A: Voltage | 78.125 | 1 | 78.125 | 41.48 | 0.0013 |
| AA | 27.0833 | 1 | 27.0833 | 14.38 | 0.0127 | |
| Total error | 9.41667 | 5 | 1.88333 | |||
| Diffusion Coefficient |
Factor A: Voltage | 0.0772245 | 1 | 0.0772245 | 38.10 | 0.0016 |
| Factor B: Flow Rate | 0.0218405 | 1 | 0.0218405 | 10.78 | 0.0219 | |
| AC | 0.0200223 | 1 | 0.0200223 | 9.88 | 0.0256 | |
| Total error | 0.0101347 | 5 | 0.00202693 | |||
⁎Degree of freedom (n-1). ⁎⁎The tabulated value of F. A is the voltage factor. B is the flow rate factor. C is the drum collector speed factor.
AB, AC, and BC are the interaction terms between the factors. AA, BB, and CC are the quadratic terms of the factors.
Fig. 4: The main effects of different factors on each response variable
Effects on zeta potential
Effects on zeta potential (Y2) were investigated. The Y2 response average ranged from 11 mV in B12 to 23 mV in B15, as shown in table 2. It was found that the factor that is mainly responsible for the difference in the zeta potential of the nanofiber is the applied voltage, as displayed in the Pareto chart of Y2, fig. 3. Fig. 4 shows almost an inverse relationship between both (X1), and zeta potential (Y2). At the same level of X2 and X3 in B3 and B9, the rise in the voltage from 12 KV to 20 KV was correlated with the lowering in the zeta potential average from 20 mV to 12 mV.
Effects on the diffusion coefficient
Effects on the diffusion coefficient (Y3) were studied. The Y3 response average ranged from 0.295 µm2/s in B8 to 0.650 µm2/s in B7, as shown in table 2. It was found that the applied voltage and injection flow rate factors are responsible for the difference in the diffusion coefficient of the nanofiber, as displayed in the Pareto chart of Y3, fig. 3. Fig. 4 shows almost an inverse relationship between both (X1) and (X2), and the diffusion coefficient (Y3). At the same level of X2 and X3 in B3 and B9, the rise in the voltage from 12 KV to 20 KV was correlated with the decrease in diffusion coefficient average from 0.604 µm2/s to 0.356 µm2/s. Also, at the same level of X1 and X3 in B5 and B6, the rise in the flow injection rate from 1.2 ml/h to 1.8 ml/h correlated with the decrease in diffusion coefficient average from 0.524 µm2/s to 0.367 µm2/s.
Prediction and fabrication of the optimized nanofiber formula
Estimated response surface
Estimated response surfaces (fig. 5) were constructed. They showed the optimum values of the three influential factors included in the formulation process of the nanofibers, which can minimize the diameter and maximize both the nanofiber's zeta potential and diffusion coefficient. It was found that for the minimum diameter, the optimum values are 12.3 KV for the applied voltage, 1.2 ml/h for the injection flow rate, and 583.029 rpm for the speed of the rotary collector, while for the maximum zeta potential, it is 13.17 KV for the applied voltage, 1.60784 ml/h for the injection flow rate, and 200 rpm for the speed of the rotary collector. For maximum diffusion coefficient, these are the values of 12.0149 KV for voltage, 1.22 ml/h for the injection flow rate, and 980 rpm for the speed of the rotary collector that gives the maximum response.
Multiple response optimization was developed to obtain optimized nanofiber experimental conditions that satisfy the intended purpose. From the desirability study and multiple response optimization, it was concluded that the optimum conditions values that can compromise all factors to produce the optimum nanofiber formula via minimizing diameter and maximizing both zeta potential and diffusion coefficient of nanofibers were found to be 12.9 KV for voltage, 1.3 ml/h for a flow rate, and 920 rpm for the speed of the rotary collector (fig. 5).
Fig. 5: The estimated response surface shows the optimum values of all factors for each response variable, and desirability shows the composition of the optimized prepared formula
Verification of the optimized nanofiber formula
All actual responses of the optimum prepared nanofiber were measured (three measurements for each response variable) and evaluated against the predicted responses using the equations:
.
The expected responses of the optimized nanofiber were 503.417 nm, 20.2083 mV, and 0.699958 µm2/s for diameter, zeta potential, and diffusion coefficient, respectively.
The observed responses of the optimized nanofiber were found to be 511±0.66 nm, 20.701±0.20 mV, and 0.72±0.03 µm2/s for diameter, zeta potential, and diffusion coefficient, respectively.
The optimized formula's residual values (difference between predicted and observed values) were 7.583 for diameter, 0.4927 for zeta potential, and 0.020042 for the diffusion coefficient (table 4).
Evaluation of the prepared nanofiber formulas
FTIR spectra
As shown in fig. 6, The nanofibrous scaffolds of both preliminary and optimized formulas exhibit distinct peaks in their FTIR spectra. The polyurethane nanofiber had identical characteristics in all scaffolds, including a carbonyl peak at 1705 cm-1, C-C absorption at 1533 cm-1, C-O stretching at 1112 cm-1, and CH-stretching vibrations at 779 cm-1, indicating the presence of substituted benzene molecules [38]. The heightened peaks validate the successful amalgamation of collagen and phytoceramides. In the polyurethane-collagen spectrum, N-H and C-H stretching peaks at 3310 cm-1and 3062 cm-1 for amide A and amide B indicated the presence of amino functional groups and hydrogen bonding inside the protein's N-H group. The C=O stretching was observed at a wavenumber of 1630 cm-1, while the amide band II, associated with the C-N vibration, was detected at 1533 cm-1 [39]. The addition of phytoceramide caused the spectrum area between 3100 cm-1and 2800 cm-1to show two strong bands at around 2943 cm-1and 2865 cm-1, indicating the stretching vibrations of methylene (n(CH2)). The frequency of these bands is associated with alterations in molecular structure. The polyurethane/collagen/phytoceramides nanofibers peaked around 1600 cm-1, corresponding to the amide I vibration of the carbonyl group (υ C=O). Another peak around 1533 cm-1 is attributed to the amide II mode involving the in-plane bending of the N-H bond (δ (N-H)) and the stretching of the C-N bond (υ C-N) [40]. It appears that the amide C=O group forms strong hydrogen bonds with the amide I band due to its placement. All of these findings point to phytoceramides being integrated and held on by the scaffolds.
Wetting properties
A scaffold's wettability is an essential bio-functional feature since it facilitates the adsorption of biomolecules and proteins onto the surface of biomaterials. Protein adsorption calls for a well-balanced degree of hydrophilicity for adequate cell adhesion and colonization. Notably, cell adhesion, proliferation, and migration are all affected by the hydrophilicity of scaffolds. Adding phytoceramide lowers the water contact angle for polyurethane and collagen nanofibers [41].
The water contact angle (WCA) is a measure of wettability. Adding phytoceramides to the polyurethane/collagen mixture decreases the water contact angle and wettability. The water contact angles were found to be 58.3±4.16 and 56.7±3.58 for the preliminary and optimized nanofiber formulas, respectively.
Table 4: Predicted, observed values, the residuals, and the prediction error percentage for the optimized nanofiber
| Response variables | Predicted⁎ | Observed⁎⁎ (Experimental) | Residual ⁎⁎⁎ | Prediction error (%) ⁎⁎⁎⁎ |
| Diameter (nm) | 503.417 | 511±0.66 | 7.583 | 1.506306 |
| Zeta Potential (mV) | 20.2083 | 20.701±0.20 | 0.4927 | 2.438107 |
| Diffusion Coefficient (µm2/s) | 0.699958 | 0.72±0.03 | 0.020042 | 2.863315 |
⁎Value predicted by design. ⁎⁎Observed data are expressed as the mean±standard deviation of three experiments (n = 3). ⁎⁎⁎Residual = predicted value – mean of the observed values. ⁎⁎⁎⁎Prediction error (%) = (predicted value − observed values average)/predicted value × 100%.
Fig. 6: FTIR spectra of preliminary and optimized formulas
Cell viability
The cytocompatibility assay was performed on the HSF cell line. The cell viability values measured for all scaffolds showed that nanofibrous scaffolds exhibited relatively high cell viability, with an estimated value of almost 100% for both optimized and preliminary nanofiber formulas at a concentration of 312.5 ng/ml. In contrast, at a higher concentration of 625 ng/ml, the cell viability dropped to 96.8±3.8% and 95.1±4.5% for the optimized and preliminary nanofiber formulas, respectively. Upon reaching the concentration of 2500, cell viability became 70.2±2.9% for the optimized nanofiber formula and 70.9±5.3% for the preliminary nanofiber formula. Based on the findings, it can be concluded that the prepared formulas are safe (non-toxic) and compatible with cells.
Antioxidant activity
During the inflammatory phase of wound healing, the skin produces a substantial quantity of reactive oxygen species (ROS) as a result of injury. This process induces cellular damage, resulting in the degradation of proteins, lipids, and nucleic acids, finally culminating in cellular demise. Consequently, the process of wound healing is interrupted. Antioxidants can greatly assist in enzymatic repair and enhance metabolism. Multiple studies have shown evidence supporting the reliability and credibility of nanofibers as a potent source of antioxidants. This study aimed to evaluate the efficacy of nanofibrous membranes in eliminating free radicals by conducting a DPPH-free radical scavenging experiment. Both preliminary and optimized nanofiber formulas showed almost the same effectiveness in scavenging DPPH radicals at a rate of 84.5%.
In vitro anti-inflammatory potency
Wound dressing can be categorized into three distinct phases: (I) inflammation, (II) neo-tissue development, and (III) tissue remodelling [42–45]. Hence, the utilization of anti-inflammatory drugs is essential to accelerating the wound healing process. Ceramide has an impact on the different metabolic and pathophysiological pathways affected by these conditions and events in an organism. These pathways are regulated by pro-inflammatory cytokines such as TNF-α, interleukin 1 (IL-1), and IL-6 [46]. The anti-inflammatory efficacy of nanofiber formulations was evaluated in comparison to the standard diclofenac sodium treatment using a denaturation of protein assay. The standard drug showed the highest anti-inflammatory effectiveness (96.3±1.5%), followed by the optimized nanofiber formula (82.8 ±2.6%) and the preliminary nanofiber formula (80.8±2.1%).
Wound healing assay
To attain the desired level of wound closure in human skin fibroblast (HSF) cells, the nanofibrous scaffolds were put in the corresponding wells. Graphs were created at 0 h, 24 h, and 48 h to illustrate the length (measured in micrometers) of the wound area. The performance of the scaffolds was tested by examining photographs, as shown in fig. 7. After the investigation was completed, the width of the wound area was calculated using the data collected.
The results show a gradual decrease in wound width, starting from 399.6±5.8 μm and reaching 95.8±3.4 μm at 48 h, with a wound closure rate of approximately 76% for the preliminary formula. The optimized formula showed a closure rate of about 79%. The wound width decreased to 85.5±6.2 μm after 48 h.
Fig. 7: The dynamic changes in wound area width over 48 h contrast the effects of preliminary and optimized nanofiber webs
Stability study
Table 5 showed that the results of the diameter and diffusion coefficient response variables obtained from stability studies performed at accelerated conditions (40 ○C and 75% RH) revealed significant differences between the months' (0, 1, 2, and 3) study points. The results of the same response variables obtained from stability studies performed at ambient temperature conditions showed no significant differences between study points (table 6).
There was no noticeable change in zeta potential findings obtained from statistical comparisons performed at different points' results at both normal ambient and accelerated conditions.
It was concluded, according to two-way ANOVA statistical analysis, that the results of the preliminary formula before optimization were significantly different from the results of the optimized one concerning diffusion coefficient at accelerated conditions (40 ○C and 75% RH).
From all these findings, a separate statistical analysis (one-way ANOVA) on the results obtained from diameter and diffusion coefficient measurements for the preliminary and optimized formulas at accelerated conditions (40 ○C and 75% RH) was recommended for the assessment of stability (tables 7 and 8).
Table 5: Two-way ANOVA statistical analysis within a 95% confidence interval on response variable data obtained at an accelerated stability study under 40 ○C and 75% RH conditions
| Two Way ANOVA | Dependent variable: responses | |||||
| Response variable⁎ | Source | Type III sum of squares | df⁎⁎ | Mean square | F⁎⁎⁎ | P-value |
| Diameter | Between Months | 3872.333 | 3 | 1290.778 | 15.321 (3.245⁎⁎⁎⁎) | 0.000 |
| Between Formulae | 130.667 | 1 | 130.667 | 1.551 (4.494 ⁎⁎⁎⁎) | 0.231 | |
| Error | 1348.000 | 16 | 84.250 | |||
| Zeta Potential |
Between Months | 0.391 | 3 | 0.130 | 0.213 (3.245⁎⁎⁎⁎) | 0.886 |
| Between Formulae | 0.095 | 1 | 0.095 | 0.155 (4.494 ⁎⁎⁎⁎) | 0.699 | |
| Error | 9.796 | 16 | 0.612 | |||
| Diffusion Coefficient |
Between Months | 0.050 | 3 | 0.017 | 29.269 (3.245⁎⁎⁎⁎) | 0.000 |
| Between Formulae | 0.031 | 1 | 0.031 | 54.219 (4.494 ⁎⁎⁎⁎) | 0.000 | |
| Error | 0.009 | 16 | 0.0006 | |||
⁎For each response variable, three experiments (n = 3) were performed for each formula at each study point (month). ⁎⁎Degree of freedom (n-1). ⁎⁎⁎The calculated value of F. ⁎⁎⁎⁎The tabulated value of F.
Table 6: Two-way ANOVA statistical analysis within a 95% confidence interval on response variable data obtained under ambient temperature conditions
| Two way ANOVA | ||||||
| Dependent variable: responses | ||||||
| Response variable⁎ | Source | Type III sum of squares | df⁎⁎ | Mean square | F⁎⁎⁎ | P-value |
| Diameter | Between Months | 125.792 | 3 | 41.931 | 0.363 (3.245⁎⁎⁎⁎) | 0.781 |
| Between Formulae | 2.042 | 1 | 2.042 | 0.018 (4.494 ⁎⁎⁎⁎) | 0.896 | |
| Error | 1850.667 | 16 | 115.667 | |||
| Zeta Potential |
Between Months | 0.077 | 3 | 0.026 | 0.050 (3.245⁎⁎⁎⁎) | 0.985 |
| Between Formulae | 1.279 | 1 | 1.279 | 2.505 (4.494 ⁎⁎⁎⁎) | 0.133 | |
| Error | 8.167 | 16 | 0.510 | |||
| Diffusion Coefficient |
Between Months | 0.0012 | 3 | .000405 | 0.590 (3.245⁎⁎⁎⁎) | 0.630 |
| Between Formulae | 1.204E-5 | 1 | 1.204E-5 | 0.018 (4.494 ⁎⁎⁎⁎) | 0.896 | |
| Error | 0.011 | 16 | 0.000687 | |||
⁎For each response variable, three experiments (n = 3) were performed for each formula at each study point (month). ⁎⁎Degree of freedom (n-1). ⁎⁎⁎The calculated value of F. ⁎⁎⁎⁎The tabulated value of F.
Table 7: One-way ANOVA statistical analysis within a 95% confidence interval on response variable data obtained from the preliminary formula at an accelerated stability study under 40 ○C and 75% RH conditions
| One way ANOVA | ||||||
| Dependent variable: responses | ||||||
| Response variable⁎ | Source | Sum of squares | df⁎⁎ | Mean square | F⁎⁎⁎ | P-value |
| Diameter | Between Groups ⁎⁎⁎⁎ | 3708.667 | 3 | 1236.222 | 13.222 (4.07⁎⁎⁎⁎⁎) | 0.002 |
| Within Groups | 748.000 | 8 | 93.500 | |||
| Total | 4456.667 | 11 | ||||
| Diffusion Coefficient |
Between Groups⁎⁎⁎⁎ | 0.064 | 3 | 0.021 | 27.574 (4.07⁎⁎⁎⁎⁎) | 0.000 |
| Within Groups | 0.006 | 8 | 0.001 | |||
| Total | 0.070 | 11 | ||||
⁎For each response variable, three experiments (n = 3) were performed at each study point (month). ⁎⁎Degree of freedom (n-1). ⁎⁎⁎The calculated value of F. ⁎⁎⁎⁎Between study points (months). ⁎⁎⁎⁎⁎The tabulated value of F.
Table 8: One-way ANOVA statistical analysis within a 95% confidence interval on response variable data obtained from the optimized formula at an accelerated stability study under 40 ○C and 75% RH conditions
| One way ANOVA | ||||||
| Dependent variable: responses | ||||||
| Response variable⁎ | Source | Sum of squares | df⁎⁎ | Mean square | F⁎⁎⁎ | P-value |
| Diameter | Between Groups ⁎⁎⁎⁎ | 786.000 | 3 | 262.000 | 3.493 (4.07⁎⁎⁎⁎⁎) | 0.070 |
| Within Groups | 600.000 | 8 | 75.000 | |||
| Total | 1386.000 | 11 | ||||
| Diffusion Coefficient |
Between Groups ⁎⁎⁎⁎ | 0.004 | 3 | 0.001 | 3.770 (4.07⁎⁎⁎⁎⁎) | 0.059 |
| Within Groups | 0.003 | 8 | 0.000 | |||
| Total | 0.007 | 11 | ||||
⁎For each response variable, three experiments (n = 3) were performed at each study point (month). ⁎⁎Degree of freedom (n-1). ⁎⁎⁎The calculated value of F. ⁎⁎⁎⁎Between study points (months). ⁎⁎⁎⁎⁎The tabulated value of F.
DISCUSSION
The current work aims to develop and optimize a wound-healing nanofiber formula using Box-Behnken statistical design. A combination of polyurethane/collagen/phytoceramides is employed for tissue engineering using the electrospun technique, which includes many instrumental factors affecting the performance of the prepared nanofiber formulas. Electrospun parameters must be controlled to ensure the effectiveness of the synthesized nanofibers.
The factors involved in the establishment of the box-behnken design were carefully selected concerning prior reports [27, 29]. The speed of the rotary collector affects the surface area of the nanofiber and thus alters the values of both the diameter and diffusion coefficient [26]. Also, previous studies show the effect of the applied voltage of the electrospun instrument on zeta potential, diameter, and diffusion coefficient [23–25]. The flow rate of the injection can modify the morphology and porosity of nanofibers and hence their diameter and diffusion coefficient [21, 22].
Results in fig. 3 and table 3 show that the diameter is significantly affected by the voltage, injection flow rate, and speed of rotary collector factors. In contrast, the zeta potential is affected significantly by voltage. Finally, the voltage and flow rate significantly affect the diffusion coefficient. The results are well supported by other relevant literature.
The findings showed that higher voltage resulted in a reduction in both the zeta potential and diffusion coefficient. This inverse relationship can be attributed to the increased elongation of the fibers at higher voltages, leading to a more compact structure and reduced surface charge. Similar observations were made by Bhardwaj and Kundu [47], who reported that increased electrospinning voltage led to fibers with reduced zeta potential.
Better drug release and cellular interactions are probably made possible by the increased diffusion coefficient and zeta potential, which also likely contribute to the increased bioactivity. These findings are consistent with those of Chenxi Li et al. [48], who discovered that nanofibers with higher zeta potential and diffusion coefficients exhibit greater biological effectiveness.
The design of the experiment effectively identified the critical parameters affecting the nanofiber characteristics. The optimal conditions were determined to be 12.9 KV for the applied voltage, 1.3 ml/h for the injection flow rate, and 920 rpm for the speed of the rotary collector. These conditions produced nanofibers with a diameter of 511±0.66 nm, a zeta potential of 20.701±0.20 mV, and a diffusion coefficient of 0.72±0.03 µm²/s, with minimal deviations between predicted and observed values. Results represented in table 4 show slight differences (error) between observed and predicted design values, indicating that the corresponding design is valuable for optimizing the nanofiber and that the optimized prepared nanofiber achieves the desired diameter, zeta potential, and diffusion coefficient.
The physicochemical criteria examination regarding wetting properties shows that the water contact angle of the optimized nanofiber formula is 56.7±3.58, which is lower than that of the preliminary formula before optimization (58.3±4.16), indicating that the hydrophilicity of the scaffolds was improved by the optimization process [49]. The reduced water contact angle between the optimized nanofibers and the initial formulations demonstrated the enhanced hydrophilicity of the optimized nanofibers. Because it encourages improved cell adhesion, proliferation, and migration on the nanofiber scaffolds, enhanced hydrophilicity is advantageous for wound healing application.
The FTIR spectra display distinct peaks, such as the carbonyl peak at 1705 cm-1, the N-H and C-H stretching peaks at 3310 cm-1 and 3062 cm-1, and the methylene stretching vibrations around 2943 cm-1and 2865 cm-1. These peaks provide evidence of the composite nanofibers' chemical integrity and compatibility. These results are consistent with earlier research by Chenghao Yu et al. [50], which showed that collagen and polyurethane-integrated nanofibers have comparable spectrum characteristics.
Furthermore, biomedical effectiveness studies, including both in vitro anti-inflammatory potency and wound healing assays, show significant improvement in results for the optimized formula compared with the preliminary suggested formula before optimization. Anti-inflammatory potency increased from 80.8 ±2.1% in the preliminary nanofiber formula to 82.8±2.6% in the optimized nanofiber formula. The optimized formula also shows a closure rate of about 79% after 48 h, while that of the preliminary formula before optimization was 76%. Results support the idea that the corresponding experimental design can improve the efficacy of the nanofiber formula for wound healing and tissue engineering purposes.
Also, stability study data represented in tables 7 and 8 indicates that only the optimized formula shows no significantly different results (p>0.05) between the successive month's points for all response variables, suggesting that it is the formula that has the most stable results; consequently, the optimization process can enhance the stability of the preliminary dosage form.
All these findings suggest that the established design succeeded in the optimization process. The results demonstrate the efficacy of the Box-Behnken design approach in the enhancement of both the effectiveness and stability of the nanofiber formula.
CONCLUSION
Polyurethane-collagen-phytoceramide nanofibers proved highly efficacious in the wound healing process. The nanofiber preparation includes parameters that significantly affect the shape and morphology of the nanofiber and, consequently, its effectiveness and stability. Factors that mainly affected the response variables of the nanofibers were the applied voltage, injection flow rate, and the speed of the rotary collector. Optimization of the nanofibers via minimizing their diameter and maximizing both the zeta potential and diffusion coefficient of nanofibers was achieved. The optimum values of factors were found to be 12.9 KV for voltage (X1), 1.3 ml/h for flow rate (X2), and 920 rpm for the speed of the rotary collector (X3). The minimal differences obtained between the design predicted values and the observed values of the optimized nanofiber responses, satisfactory physicochemical characteristics, high biomedical effectiveness, and results of the stability study indicate that the corresponding design succeeded in optimizing the preparation process and that the optimized nanofiber verified the desired diameter, zeta-potential, and diffusion coefficient. Thus, the optimization process achieved the intended goal.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
Tassneim M. Ewedah conducted the experiments and participated in the article's writing. Mohamed El-Nabarawi wrote the paper's outline and contributed to preparing and reviewing the article. Mahmoud H. Teaima was a research guide and participated in the review of the article. Sammar Fathy Elhabal participated in the discussion of the results and review of the article. Kamel R. Shoueir conducted the experiments and participated in the discussion of the results. Abdallah M. Hamdy participated in the design, analysis, and interpretation of the data. Ahmed Abdalla participated in the article's writing and discussion of the results.
CONFLICT OF INTERESTS
The authors have no conflicts of interest
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