Wound Healing Efficacy of Rosuvastatin Transethosomal Gel, I Optimal Optimization, Histological and In Vivo Evaluation

This study aimed to make a formulation and statistical optimization of transethosomal formulations of rosuvastatin (ROS) to enhance its topical wound healing efficiency. Design-Expert® software was used to employ I optimal design. The formulation variables in the study were surfactant concentration (%w/v), ethanol concentration (%w/v) and surfactant type (span 60 or tween 80), while the dependent responses were entrapment efficiency percent (EE%), vesicle size (VS) and zeta potential (ZP). The numerical optimization process employed by the design expert software resulted in an optimum formula composed of 0.819439 (%w/v) span 60, 40 (%w/v) ethanol and 100 mg lecithin with a desirability of 0.745. It showed a predicted EE% value of 66.5517 vs. 277.703 nm and a ZP of −33. When it was prepared and validated, it showed less than a 5% deviation from the predicted values. The optimum formula was subjected to further characterizations, such as DSC, XRD, TEM, in vitro release, the effect of aging and wound healing efficiency. The DSC thermogram made a confirmation of the compatibility of ROS with the ingredients used in the formulation. XRD showed the encapsulation of ROS in the transethosomal vesicles. The TEM image pointed out the spherical nature of the nanovesicles with the absence of aggregation. Additionally, the optimum formula revealed an enhancement of drug release in comparison with the drug suspension. It also showed good stability for one month. Furthermore, it revealed good wound healing efficiency when compared with the standard silver sulphadiazine (1% w/w) ointment or the drug-loaded gel, which could be related to the enhanced penetration of the nanosized vesicles of TESMs into the skin, which enhances the wound healing process. So, it could be regarded as a promising carrier of ROS for the treatment of chronic wounds.


Introduction
Wounds are considered an important risk factor for morbidity all over the world due to microbial infections [1]. Wound healing is a complex process of multiple phases, including homeostasis, inflammatory reactions, cell proliferation and tissue remodeling [2]. The delay Table 1. I optimal design for optimization of ROS-loaded TESMs.

Preparation of ROS Transethosomal Formulations
TESMs were prepared by applying the thin film hydration technique [20], where lecithin, ROS and surfactant were dissolved in 10 mL chloroform-methanol mix at a ratio of 2:1 in a round bottom flask, followed by evaporating the organic solvent using a rotary evaporator (Buchi Rotavapor R-200, Switzerland) by applying a vacuum at a temperature of 60 • C at 90 rpm until the formation of a thin film. Then, 10 mL of water containing the calculated amounts of ethanol was used to hydrate the film at 60 • C, which was higher than the transition temperature of the lipid phase (Tc) [28].

Evaluation of ROS Transethosomal Formulations 2.4.1. Measurement of Entrapment Efficiency (EE%)
A cooling centrifuge (SIGMA 3-30 K, Sigma, Steinheim, Germany) was used to separate transethosomal vesicles from the un-entrapped ROS by centrifugation at 17,000 rpm for 1 h at 4 • C [32]. Then, a UV spectrophotometer (Shimadzu UV-1800, Kyoto 604-8511, Japan) was used to quantify the ROS concentration in the supernatant after being suitably diluted. The measurements were performed at the predetermined λ max (245 nm). The method was validated in terms of linearity within the concentration, which ranged from 2 to 16 µg/mL (R 2 of 0.9995).
The EE% was calculated by the use of the following equation [33]: where EE% is the percent of entrapment efficiency, FD is the amount of free drug, and TD is the amount of the total drug.

Measurement of Vesicle Size (VS), Polydispersity Index (PDI) and Zeta Potential (ZP)
The measurements of the VS, PDI and ZP of the prepared ROS-loaded TESMs were performed using a Zetasizer Nano ZS instrument (Malvern Instruments, Worcestershire, UK) at 25 • C after being suitably diluted with distilled water [33,34]. Each measurement was performed three times.

Statistical Analysis, Optimization and Validation
The studied responses were subjected to analysis using a factorial analysis of variance (ANOVA) applying Design Expert ® software. The optimum formula with the highest EE% and ZP, and the smallest VS was selected using a desirability function. Then, it was prepared and subjected to evaluation in terms of EE%, VS and ZP to verify the validity of the applied statistical models by calculating the percentage relative errors between the predicted values and the measured results by applying the following equation [34,35].
2.6. Evaluation of the Optimum ROS Transethosomal Formula 2.6.1. Differential Scanning Calorimetry (DSC) Pure ROS, a physical mixture of lecithin, span 60, and ROS, and the optimum formula were subjected to a DSC analysis by means of a differential scanning calorimeter (DSC N-650; Scinco, Italy). About 5 mg of each sample were placed in the apparatus's aluminum pan, followed by heating at a rate of 10 • C per minute until 200 • C underflows of inert nitrogen.

X-ray Diffraction Study (XRD)
Ultima IV Diffractometer (Rigaku Inc. Tokyo, Japan at College of Pharmacy, King Saud University, Riyadh, Saudi Arabia) was used to measure the X-ray diffraction patterns of pure ROS, physical mixture of lecithin, span 60 and ROS, and the optimum formula. They were subjected to scanning at a rate of 10 • per minute of speed in the range from 0-60 • (2θ).

Transmission Electron Microscopy (TEM)
The optimum formula morphology was visualized using a transmission electron microscope (TEM; JEOL JEM-1010, Tokyo, Japan). The samples were subjected to suitable dilutions. Then, they were placed on a carbon-coated copper grid. After that, they were coated with 2% w/v phosphotungstic acid, followed by keeping them in the air for 5 min to be dried. Then, they were imaged using TEM operated under an acceleration voltage of 80 kV [36] and X80000 power of magnification at room temperature.

In Vitro Release
The release of ROS from the optimum ROS-loaded TESMs formula in comparison with the ROS-loaded TESMs gel and drug suspension was studied by introducing the equivalent of 5 mg ROS of each in the dialysis bags, followed by suspending each in a dissolution medium of 250 mL (phosphate buffer pH (7.4)) [37] in the dissolution apparatus (Pharm Test, Hainburg, Germany) at 37 • C and stirring at 100 rpm. Samples of 5 mL were withdrawn from the dissolution media at 1, 2, 3, 4, 5 and 6 h and instantaneously replaced with a fresh medium of an equal volume. After that, the concentration of ROS in the gathered samples was determined using a UV spectrophotometer at 245 nm. The percent of ROS released at different time points was calculated as follows: [38] Where 1.
Qn: Cumulative percent of ROS released 2.
Cn: Concentration of ROS in the dissolution medium at the n th sample 3.
Vr: Volume of dissolution medium 4.
Vs: Volume of sample 5.
Ci: The summation of the concentrations measured previously The percentage of ROS released (Q n ) at various time points was plotted vs. the corresponding time to obtain the release profile of the optimum ROS-loaded transethosomal formula in comparison with the drug suspension.

Effect of Aging
The stability of the optimum ROS-loaded transethosomal formula was determined as a function of time regarding EE%, VS and ZP after placing the formulation in an air-tight vial and keeping it at 4 • C and away from light for one month [39].

Preparation of ROS Transethosomal Gel
The optimum formula was incorporated in a gel base to be applied topically in the in vivo studies. The polymer of choice for the gelling process was hydroxypropyl methylcellulose (HPMC, K4M) at a concentration of 2.5%. Gel preparation was performed by dispersing 0.25 g HPMC in 10 mL distilled water while stirring at 1000 rpm until the formation of a homogenous system. The optimum TESM formula was subjected to ultracentrifugation followed by dispersing the residue (ROS-loaded TESMs) in the gel base to obtain a final formulation with 1% ROS concentration [32].

Grouping and Dosing of Animals
Male rats weighing 120 ± 20 g were separated into five groups. Each group contained six animals. The first group contained control animals (normal animals without wound induction). The second group contained animals with a wound who did not receive any treatment. The third group took standard silver sulphadiazine (1% w/w) ointment as a treatment. The fourth group received drug-loaded gel (1% ROS in 2.5% HPMC, K4M). Finally, the last group received the optimum ROS transehosomal gel formula. Before the start of the study, the animals were supplied with standard food and water ad libitum and acclimatized to the laboratory conditions.

Experimental Design
The creation and excision of wounds on the rats were initiated by making anesthesia using an IV injection of ketamine (120 mg/kg body weight) followed by shaving the mice's backs. Then a scalpel and sharp scissors were used to create the wound on the sides of the central trunk, followed by sterilization using ethanol and removing the skin from the marked area to obtain a wound of 135 mm 2 at maximum. Afterward, wound cleaning was performed using a cotton swab soaked in saline, followed by the placing of the animals in individual cages and the gentle application of the different treatments 24 h after wound induction once per day by covering the wound until complete healing. A transparent ruler was used to measure each animal's wound diameter at 0, 7, 14 and 21 days on a weekly basis until epithelialization and the recording of complete wound closure. The wound area gave an indication of the activity of wound healing, in addition to the wound contraction rate percent [40]. The following equation was used to calculate the percent wound contraction: %wound contraction = Initial size of the wound − Wound size in a specific day Initial size of the wound × 100 Samples of skin tissue (3-5 cm) from different animal groups were instantly dipped in a suitable amount of 10% formalin.
Wound healing models cause moderate to severe pain. Multimodal strategies and therapy paved the way for modern robotic surgeries to take place so as to reduce the need for frequent doses of painkillers, faster recovery and the complete healing of wounds to prevent the nightmare of chronic pain. This became the key interest in our study: to look out for a speedy recovery in all ways from the new drug [41].

Histological Study
To compare the histological effect of the investigated material on wound healing, we used two stains on formalin-fixed paraffin wax as a fixed representative and a suitable size of skin tissue biopsies. Thus, representative wound skin tissue samples with a thickness from 3-5 cm from the five animal groups were instantly immersed in a suitable amount of 10% formalin and prepared in an automatic tissue processing machine (ASP300s, Leica Bio systems, Buffalo Grove, IL, USA), followed by impeding them in paraffin wax blocks. Then 5 µ thick sections were prepared using a rotary microtome (SHUR/Cut 4500, TBS, Durham, NC, USA) [42]. Two sections of each block were taken for staining; one was stained with the hematoxylin and eosin (H&E) technique for general tissue appearance staining, and the second was stained with the Masson trichrome technique (MT) for connective tissue fibers, mainly collagen, which takes blue color [43,44]. The hematoxylin and eosin method was performed by the following descriptions of Bancroft and Layton [44]. The Masson trichrome techniques were completed according to Hamad et al. [43].

Measurement of EE%
The EE% of the prepared transethosomal formulations varied between 45.2 ± 2.58 and 87.3 ± 3.62, as shown in Table 2   The linear model was the most suitable one fitted to EE% data (p-value < 0.0001), where the lack of fit is non-significant (p-value 0.132), and the difference between the adjusted and predicted R 2 was small (less than 0.2), which points out that the model is valid [34]. The adequate precision was high, 88.1062 (greater than four), as shown in Table 3. This referred to the ability of the model to navigate the design space [45,46]. The linear model was the most suitable one fitted to EE% data (p-value < 0.0001), where the lack of fit is non-significant (p-value 0.132), and the difference between the adjusted and predicted R 2 was small (less than 0.2), which points out that the model is valid [34]. The adequate precision was high, 88.1062 (greater than four), as shown in Table 3. This referred to the ability of the model to navigate the design space [45,46]. The effect of the independent variables on EE% was shown in the following equation: It can be concluded from the ANOVA analysis shown in Table 4 that all three independent variables namely, surfactant concentration %w/v (X1), ethanol concentration %v/v (X2) and surfactant type, have a significant effect on EE% values with p-values < 0.0001. Increasing both surfactant and ethanol concentrations led to a significant decrease in EE%, as indicated by the negative sign of their coefficients in the correlation equation.
Regarding the effect of surfactant concentration on EE%, the decrease in EE% with the increase in surfactant concentration could be related to increasing the membrane permeability of the vesicles, which resulted from the arrangement of the surfactant molecules within the lipid bilayer structure of the vesicles, which led to the generation of pores, within the membrane resulting in increasing its fluidity which increased leakage of the entrapped drug [47].
Concerning the effect of ethanol concentration on EE%, there was a decrease in the EE% with the increase in the concentration of ethanol, which could be explained by the solubilization of the drug in ethanol in addition to the effect of ethanol on the vesicle's membrane, which led to enhanced fluidity and permeability with the subsequent loss of the drug from it [48]. Our findings complied with those published by Abdulbaqi et al. [49].
For the effect of surfactant type on EE%, it was obvious that the EE% was higher in formulations containing span 60 in comparison w those prepared with tween 80. These results were in agreement with the results published by Aboud et al. [50] and could be referred to as the hydrophilic-lipophilic balance (HLB) values of span 60 and tween 80, which were 4.7 and 15, respectively [28,50,51]. Surfactants with low HLB are lipophilic and would prefer the entrapment of lipophilic drugs [52]. This explained the increased EE% of ROS, which is a lipophilic drug [16], in formulations containing span 60 than those containing tween 80. In addition, there was some kind of interaction between the hydrophobic alkyl chain of span 60 and the hydrophobic domain in the vesicles, which results in more condensed layers and so prevents the leeching of drugs from the vesicles [53]. Contrarily, surfactants with high HLB, such as tween 80, are more hydrophilic and form vesicles with less rigid membranes, which is related to the larger polar head groups in addition to increasing the solubilization of the drug in the aqueous medium during preparation, which led to lowering the EE% of ROS [54].

Measurement of VS, PDI and ZP
The VS of the prepared transethosomal formulations lay between 191.4 ± 7.84 to 372.6 ± 12.84 nm, as shown in Table 2. The effects of the surfactant concentration w/v% (X1) and ethanol concentration v/v% (X2) on VS are shown in Figures 1B and 2B.
The most appropriate model to be fitted to VS data was the two-factor interaction (p-value < 0.0001) with a non-significant lack of fit (p-value 0.154) and a small difference between the adjusted and predicted R 2 (less than 0.2), which ensures the validity of the model [34]. The adequate precision was high, 65.0316 (greater than 4); this pointed out the ability of the model to navigate the design space [45,46], as shown in Table 3.
The effect of the studied factors on VS was refereed in the following equation:  (6) It was obvious from ANOVA analysis, as seen in Table 4, that surfactant concentration w/v% (X1), ethanol concentration v/v% (X2) and surfactant type all have a significant effect on VS values with (p-values < 0.0001).
Increasing both surfactant and ethanol concentrations resulted in a significant decrease in VS, as shown by the negative sign of their coefficients in the correlation equation. The decrease in VS with the increase in surfactant concentration could be related to the softening of the membrane and increased elasticity, which results in an increased reduction ability [55]. Our results were in agreement with the results obtained by Chen et al. [56]. It was also noted from the results that the PS was in accordance with the amount of drug entrapped within the vesicles and explained that decreasing the EE% of the vesicles led to reducing its size [28].
The decrease in VS with the increase in the concentration of ethanol could be attributed to the reduction in the thickness of the membrane and due to the formation of a phase with interpenetrating hydrocarbon chains [57]. The presence of ethanol gave steric stabilization to vesicles due to imparting some negative charge, which results in enhancing the physical stability of particles and preventing their aggregation [58]. Our results met the results published by Nayak et al. [59].
Concerning the effect of surfactant type on VS, all formulae prepared using Span 60 are larger in size than those prepared using Tween 80. This could be related to the HLB values of each surfactant. Yeo et al. [60] pointed out that when the HLB value of a surfactant decreases, the VS increases, which could be linked to the reduction of the hydrophilic portion of the surfactant. That is why span 60 with the lower HLP value (4.7), as mentioned before, showed a higher VS than tween 80 with an HLP value of 15. Our results complied with results published by Rofida et al. [28].
PDI shows the variety in size between particles and is referred to by values between 0 and 1 [45]. As presented in Table 1, the PDI values of the prepared transethosomal formulations varied between 0.137 ± 0.033 and 0.501 ± 0.148; this indicated the acceptable size distribution for the prepared transethosomal formulations [45].
ZP points out the physical stability of the prepared transethosomal formulations. Where increasing the ZP value leads to increasing the repulsion forces between vesicles, which reduces their aggregation and increases system stability [61].
As seen in Table 2, the ZP of the prepared transethosomal formulations lay between −14.3 ± 2.64 and −32.7 ± 1.38 mV. This refers to the physical stability of the prepared transethosomal formulations [62]. The effects of surfactant concentration (X1) and ethanol concentration (X2) on ZP are shown in Figures 1C and 2C.
The most appropriate model to be fitted to the ZP data was the linear model (p-values < 0.0001). The adequate precision was high (46.9420), and the difference between the adjusted and predicted R 2 was less than 0.2. The effect of the studied factors on ZP could be clarified using the proceeding equation: ZP = −24.94 + 2.13 X1 + 5.12 X2 + 3.83 X3 The ANOVA analysis in Table 3 shows that both surfactant concentration (X1), ethanol concentration (X2) and surfactant type (X3) significantly affected ZP (p-values < 0.0001), where X1 and X2 significantly increased ZP absolute values. The increase of the ZP absolute value with the increase of the surfactant concentration could be related to the charge imparted by them on the vesicles' surface [47]. The increase in the ZP absolute value with the increase in the ethanol concentration could be referred to as imparting a negative charge on the vesicles ' surface, which led to electrostatic repulsion between them, avoiding their aggregation [49]. Our results were in agreement with that published by Dayan and Touitou [63].
Regarding the effect of surfactant type on ZP values, the ZP of the formulations prepared using tween 80 is less than that of those prepared using span 60, which could be explained by the higher HLP values of tween 80 in comparison with span 60. Our results complied with those published by Rofida et al. [28]. Kim et al. [64] pointed out that the HLP value of the surfactant affects the competitive adsorption of OH ions present in the hydration medium at the interface. The lower the HLP value of the surfactant, the higher the adsorption of OH ions on the nonpolar interface and the higher the ZP. The presence of (CH 2 -CH 2 -O) n in tween 80 made hydrogen bonds with water molecules, which led to the lowering of the ZP value [65].

Statistical Analysis, Optimization and Validation
A numeric analysis for the selection of the optimum transethosomal formula was made by applying Design Expert ® software, where EE% and ZP were maximized while VS was minimized. This resulted in an optimum transethosomal formula with a desirability of 0.745 ( Figure 3). Its composition was 0.819439 (w/v%) span 60, 40 (w/v%) ethanol and 100 mg lecithin. The predicted values of EE%, VS and ZP were 66.5517%, 277.703 nm and −33.3014 mV, respectively, as shown in Table 5 and Figures 3 and 4. The optimum formula was prepared and then validated, as verified in Table 4, with a percentage of relative errors of less than 5% from the predicted values produced by the design expert software, which indicated the fitness of the model [35].

Differential Scanning Calorimetry (DSC)
DSC thermograms of pure ROS, physical mixture of lecithin, span 60 and ROS and the optimum transethosomal formula are shown in Figure 5. Pure ROS exhibited an endothermic peak at 184 • C, which indicated its melting point in crystal form ( Figure 5A) [66,67]. The endothermic peak of the drug was well conserved in its physical mixture with lecithin and span 60 ( Figure 5B), with changes in the form of broadening or shifting the melt temperature. The used quantity of materials, especially in the mixtures of drugs and excipients, may have an effect on the enthalpy and shape of the peak. These minute changes in the melting endotherm of the drug may be due to making a mix between the drug and the excipients, which resulted in reducing the purity of the mixture's individual components, and this may not essentially point out a probable incompatibility [68][69][70]. In addition, drug crystallinity changes may result in shifts in the melting point [68]. So, the compatibility of ROS with the formulation excipients could be deduced. The optimum transethosomal formula ( Figure 5C) showed the absence of the drug's endothermic peak, which indicated that the drug was encapsulated and converted into an amorphous form [71].

X-Ray Diffraction Study (XRD)
The XRD spectra of pure ROS, lecithin, span 60, ROS physical mixture and the optimum formula are shown in (Figure 6). The XRD of pure ROS revealed a broad peak at an angle of 20° and sharp peaks at angles of 38° and 44°, which indicated its crystalline nature [3,12] ( Figure 6A). The XRD spectra of lecithin, span 60 and ROS physical mixture showed the appearance of a new sharp peak at an angle of 22° with the persistence of the drug peak at 20° ( Figure 6B). However, a decrease in the intensity of the drug peaks was observed in the XRD spectrum of the optimized formula ( Figure 6C), which may be due to the drug encapsulation within TESMs nanovesicles in an amorphous form. The obtained findings were in compliance with the DSC results [34].

X-ray Diffraction Study (XRD)
The XRD spectra of pure ROS, lecithin, span 60, ROS physical mixture and the optimum formula are shown in (Figure 6). The XRD of pure ROS revealed a broad peak at an angle of 20 • and sharp peaks at angles of 38 • and 44 • , which indicated its crystalline nature [3,12] ( Figure 6A). The XRD spectra of lecithin, span 60 and ROS physical mixture showed the appearance of a new sharp peak at an angle of 22 • with the persistence of the drug peak at 20 • ( Figure 6B). However, a decrease in the intensity of the drug peaks was observed in the XRD spectrum of the optimized formula ( Figure 6C), which may be due to the drug encapsulation within TESMs nanovesicles in an amorphous form. The obtained findings were in compliance with the DSC results [34].

Transmission Electron Microscopy (TEM)
Photographs of TEM revealed small vesicles of a spherical nature, as seen in Figure  7. No aggregation was observed, which points out that the dispersion was physically stable, and this might be due to the high surface ZP of the TESMs nanovesicles surfaces, which imparts repulsion forces between them [33,34].

Transmission Electron Microscopy (TEM)
Photographs of TEM revealed small vesicles of a spherical nature, as seen in Figure 7. No aggregation was observed, which points out that the dispersion was physically stable, and this might be due to the high surface ZP of the TESMs nanovesicles surfaces, which imparts repulsion forces between them [33,34].

Transmission Electron Microscopy (TEM)
Photographs of TEM revealed small vesicles of a spherical nature, as seen in Figure  7. No aggregation was observed, which points out that the dispersion was physically stable, and this might be due to the high surface ZP of the TESMs nanovesicles surfaces, which imparts repulsion forces between them [33,34].

In Vitro Release
The release profile of the optimum ROS-loaded TESMs formula compared with ROSloaded TESMs gel and ROS suspension was presented in Figure 8. There was an enhanced release of ROS from ROS-loaded TESMs compared with the drug suspension. This might be referred to as the amphiphilic properties of lecithin used in TESMs formation [72,73]. The reduction in vesicle size of the transethosomal formulation may lead to enhanced drug release [34]. Vesicle size had an effect on the release of the drug from nanovesicles, where smaller vesicles led to a higher release rate in comparison to larger-sized ones [73,74]. Additionally, ROS-loaded TESMs gel showed a slower release rate than ROS-loaded TESMs; this could be attributed to the release from transethosomal nanovesicles and diffusion of ROS through the network structures of the gel, resulting in a controlled release model for ROS-loaded TESMs gel. This result is in agreement with Zaki et al. [32].

In Vitro Release
The release profile of the optimum ROS-loaded TESMs formula compared with ROSloaded TESMs gel and ROS suspension was presented in Figure 8. There was an enhanced release of ROS from ROS-loaded TESMs compared with the drug suspension. This might be referred to as the amphiphilic properties of lecithin used in TESMs formation [72,73]. The reduction in vesicle size of the transethosomal formulation may lead to enhanced drug release [34]. Vesicle size had an effect on the release of the drug from nanovesicles, where smaller vesicles led to a higher release rate in comparison to larger-sized ones [73,74]. Additionally, ROS-loaded TESMs gel showed a slower release rate than ROSloaded TESMs; this could be attributed to the release from transethosomal nanovesicles and diffusion of ROS through the network structures of the gel, resulting in a controlled release model for ROS-loaded TESMs gel. This result is in agreement with Zaki et al. [32].

Effect of Aging
The effect of one month's storage on the stability of the optimum transethosomal formula is shown in Table 6 and Figure 9. The EE%, VS and ZP did not significantly change during the study periods (7 and 30 days), which could reflect the good stability of the optimum transethosomal formula during one month's storage at 4 °C [34].

Effect of Aging
The effect of one month's storage on the stability of the optimum transethosomal formula is shown in Table 6 and Figure 9. The EE%, VS and ZP did not significantly change during the study periods (7 and 30 days), which could reflect the good stability of the optimum transethosomal formula during one month's storage at 4 • C [34]. Wound closure was confirmed by measuring the diameter of the wounds. The group that did not receive any treatment (group 2) was used to verify the normal healing activity in the animal model. All animal groups revealed a decrease in wound area daily until the end of the study after 21 days as compared with the start date, as shown in Table 7 and Figure 10A,B. Moreover, wound epithelization time was longer in group two compared with the other treatment groups. As shown in Figure 10A,B, the group five animals, which were treated with the ROS tranethosomal gel formula, showed larger wound closure in comparison with the other groups, which could be related to the penetration of nanosized vesicles of TESMs into different skin layers, which enhances the wound healing process [18,75]. Additionally, the presence of edge activators in the composition of transethosomes enhances skin permeation by increasing the fluidity of transethosomal lipid bilayer and consequently easifying their squeezing into the skin pores [76,77]. Moreover, the high concentration of ethanol enhances skin permeation by two mechanisms: first, it interacts with lipid molecules of stratum corneum causing a change in the packing of skin lipids and consequently increasing their fluidity and permeability; second, it increases the fluidity and flexibility of transethosomal lipid bilayers and so increases their permeation through the skin [28]. Two-way ANOVA analysis showed that all the groups are significantly different from each other in wound healing activity (p-value < 0.021254), and also there is a significant difference in wound healing activity on days 7, 14 and 21 (p-value < 0.000564), as shown in Table 7. The decrease in wound size in treatment groups compared with the untreated group is shown in Figure 10A,B.

Quantification of Wound Area
Wound closure was confirmed by measuring the diameter of the wounds. The group that did not receive any treatment (group 2) was used to verify the normal healing activity in the animal model. All animal groups revealed a decrease in wound area daily until the end of the study after 21 days as compared with the start date, as shown in Table 7 and Figure 10A, B. Moreover, wound epithelization time was longer in group two compared with the other treatment groups. As shown in Figure 10A,B, the group five animals, which were treated with the ROS tranethosomal gel formula, showed larger wound closure in comparison with the other groups, which could be related to the penetration of nanosized vesicles of TESMs into different skin layers, which enhances the wound healing process [75,18]. Additionally, the presence of edge activators in the composition of transethosomes enhances skin permeation by increasing the fluidity of transethosomal lipid bilayer and consequently easifying their squeezing into the skin pores [76,77]. Moreover, the high concentration of ethanol enhances skin permeation by two mechanisms: first, it interacts with lipid molecules of stratum corneum causing a change in the packing of skin lipids and consequently increasing their fluidity and permeability; second, it increases the fluidity

The Effect of Wound Induction and Healing on Body Weight and Food and
Water Intake Figure 10C,D present the food and water intake of the rats. The daily food and water intake of the rats was significantly decreased in all the groups compared with normal animals. Food and water consumption have a direct relation to the health condition of rats; in this case, due to the wound, the animals were unable to move, resulting in a decrease in food and water consumption. The progression of wound healing is indicated by the increase in food and water intake, which is seen in the figures. Figure 10E shows the body weight changes in the groups after wound induction and wound healing in the groups. The declining pattern of body weight and regaining of body weight directly attributes to the feeding patterns of the animals. The results clearly show that there are significant body weight changes during the wound 0-7 days of wound induction and later. (14-21 days). Changes in body weight are used to assess the course of the disease and response to drug therapy. Body weight is a good indicator of pain, inflammation and stress that occurs during an injury or wound. Weight loss was observed in all the wound-induced groups, which clearly stated the alleviation of pain, inflammation and stress in the animals. Body weight changes are an important tool for indicating the feeding behavior of the animals, which has been used for a long time to quantify the chronic pain status of various animals [78][79][80].

Histological Study
Histopathological examination is another piece of evidence for the experimental wound healing activity ( Figure 11). Like our study, Zhang et al. [81], in their study of skin wounds, used both methods of H&E as well as the method of Masson trichrome to assess the efficacy of the treatment they applied. Aneesha et al. [82] used the same two histological stains to assess the wound healing of diabetic tissue samples. In addition, histopathological features were assessed for the improvement of skin tissue wound healing by using H&E and Masson trichrome in our study, like in Wahedi et al. [83].
As expected, the control group (group 1), in all three weeks, gave a normal histological appearance of the skin in both H&E stained sections ( Figure 11A) as well as MT-stained sections ( Figure 11B). Second, the toxic-induced group (group 2) showed very little improvement over the three weeks but suffered from several pathological events such as loss of epithelial tissue layer area (L), necrotic tissue area (N), hemorrhage (H), and infiltration of inflammatory cells (I). MT-stained sections of this group showed continuous suffering of the skin tissue from the decreased amount (D) of collagen fibers in connective tissue near the wound areas while a very much (V) decreased amount of collagen fibers in areas nearer to the wound. Third, the standard treated group (group 3) showed gradual improvement throughout the period of three weeks and almost normal skin tissue appearance in both H&E and MT-stained sections. Fourth, the drug-loaded gel-treated group (group 4) showed high improvement in regard to wound healing, but the tissue of the skin was not completely healed. Finally, the optimum transethosomal gel formula-treated group (group 5) showed better and highly improved skin tissue that showed complete healing and almost normal tissue appearance in both H&E and MT staining, which could be related to the reason previously discussed in Section 3.4.1. In conclusion, this histopathological experiment revealed that the optimum transethosomal gel formula-treated group exhibited different biological behavior in closing the wound area first during the first two weeks and then rapidly continued the healing of connective tissues underneath until the wound was almost normal by the end of the third week.

Conclusions
In the current study, I optimal design was employed for the optimization of ROS TESMs where EE% and ZP were maximized while VS was minimized. This resulted in an optimum formula composed of 0.819439 (%w/v) span 60, 40 (%w/v) ethanol and 100 mg lecithin with a desirability of 0.745. It showed a reasonable vesicle size of 277.703 nm, ZP of −33 and ROS entrapment efficiency of 66.5517%. This optimum formula showed spherical vesicles under TEM with no aggregates, which were confirmed by the stability study

Conclusions
In the current study, I optimal design was employed for the optimization of ROS TESMs where EE% and ZP were maximized while VS was minimized. This resulted in an optimum formula composed of 0.819439 (%w/v) span 60, 40 (%w/v) ethanol and 100 mg lecithin with a desirability of 0.745. It showed a reasonable vesicle size of 277.703 nm, ZP of −33 and ROS entrapment efficiency of 66.5517%. This optimum formula showed spherical vesicles under TEM with no aggregates, which were confirmed by the stability study for one month. It also showed enhanced drug release when compared with the drug suspension. In addition, DSC and XRD studies showed good compatibility of the drug with the excipients in the formula and revealed its encapsulation within the nanovesicles. Finally, it was subjected to a wound healing efficiency study applying an excision wound model and histology study where it showed good wound healing properties when compared with the standard silver sulphadiazine (1% w/w) ointment, and this could be related to the penetration of the nanosized vesicles of TESMs into the skin, which enhanced the wound healing process. So, it could be regarded as a promising carrier for chronic wound treatment.