Diosmin-Loaded Nanoemulsion-Based Gel Formulation: Development, Optimization, Wound Healing and Anti-Inflammatory Studies

The wound-healing process is complex and prone to interruption or failure, which can result in the development of chronic wounds that never heal. This can be overcome by seeking prompt medical attention, which will reduce the likelihood of complications and speed up the healing of the cutaneous wound. It has been established that functionalized engineered biomaterials are a possible strategy for starting skin wound care. The purpose of the current study is to develop a diosmin (DSM)-loaded nanoemulsion (NE)-based gel formulation and to investigate its wound healing and anti-inflammatory activity on rats. The DSM-loaded NEs (F1-F17) were developed and optimized with the help of Box–Behnken Design Expert. The DSM-Nes were developed using lauroglycol 90 (LG90®) as oil, Tween-80 as surfactant and transcutol-HP (THP) as co-surfactant. The optimized Nes showed globule size (41 ± 0.07 nm), polydispersity index (PDI) (0.073 ± 0.008) and percentage of entrapment efficiency (%EE) (87 ± 0.81%). This optimized DSM-loaded NEs (F1) was further evaluated and incorporated into 1% carbopol 940 gel. F1-loaded gel was then characterized for drug content, spreadability, in vitro release, wound healing, and anti-inflammatory studies. The developed gel of DSM was found to show significantly better (p < 0.05) wound-healing and anti-inflammatory activity.


Introduction
Diosmin (DSM) is a flavone glycoside derived from hesperidin, a flavanone amply found in citrus fruits [1][2][3]. It was first isolated in 1925 from the Scrophularia nodosa Lamiales that belongs to Scrophulariaceae family, indigenous to the Northern hemisphere. Its therapeutic significance was first discovered in 1969, being mainly used in Europe as phlebotonics (flavonoid-antioxidant) and vascular protector for the treatment of the foot ulcer and hemorrhoids conditions [4]. To date, many biological activities have been reported for DSM such as anti-diabetic [5], anti-cancer [6], retinal protection [7], renoprotection [8], cardiovascular protection [9,10], neuroprotective [11], anti-inflammatory [12], and antioxidant properties [13]. The anti-inflammatory effects of DSM could be due to the potent reduction in the prostaglandin and thromboxane synthesis and inhibition of leukocyte activation [14]. Gels 2023, 9, 95 3 of 20 To the best of our knowledge, there has not been any research reporting an NEs-based topical gel for wound healing and anti-inflammatory activities. Therefore, our investigation aimed to prepare and optimize DSM-loaded NEs with Box-Behnken design using an aqueous titration technique. Optimized NE was then loaded into the carbopol 940 gel and characterized according to its formulation parameters and release kinetics. We have evaluated the developed formulation and our findings are based on the in vivo excision wound models in rats followed by histopathological examinations. Figure 1 illustrates the solubility of DSM in a few selected pure oils, surfactants, and co-surfactants. The highest solubility of DSM among different tested solvents was found in LG90 (oil), surfactant (Tween-80) and co-surfactant (THP) as 30.96 ± 1.7 mg/mL, 42.08 ± 1.2 mg/mL, and 39.52 ± 2.1 mg/mL, respectively. DSM is highly hydrophobic in nature; therefore, incorporation into NEs is useful to enhance solubility [34].
To the best of our knowledge, there has not been any research reporting an NEsbased topical gel for wound healing and anti-inflammatory activities. Therefore, our investigation aimed to prepare and optimize DSM-loaded NEs with Box-Behnken design using an aqueous titration technique. Optimized NE was then loaded into the carbopol 940 gel and characterized according to its formulation parameters and release kinetics. We have evaluated the developed formulation and our findings are based on the in vivo excision wound models in rats followed by histopathological examinations. Figure 1 illustrates the solubility of DSM in a few selected pure oils, surfactants, and co-surfactants. The highest solubility of DSM among different tested solvents was found in LG90 (oil), surfactant (Tween-80) and co-surfactant (THP) as 30.96 ± 1.7 mg/mL, 42.08 ± 1.2 mg/mL, and 39.52 ± 2.1 mg/mL, respectively. DSM is highly hydrophobic in nature; therefore, incorporation into NEs is useful to enhance solubility [34].  Table 1 lists the effects of independent factors on the globule sizes of various formulations. Negative coefficients of A (oil) and C (co-surfactant) significantly affect the response Y1, showing that the mean globule size was observed to decrease with decrease in the concentration of oil and co-surfactant [36] (see Figure 2). This equation showed A, C, AC, BC, A 2 and C 2 to be significant model terms with p < 0.05. The significance of the model was demonstrated by its model F-value of 66.55 (p < 0.05). An ANOVA evaluation of the quadratic model showed that responses Y1 and Y2 were significantly fitted ( Table  2). The model performed well because the lack of fit's F-value of 0.225 suggested that it was not significant. The quadratic model has a higher R 2 (0.9884) than the other models. An appropriate signal was suggested by the sufficient precession larger than 4 (25.29) [37]. The predicted R 2 and adjusted R 2 values for response Y1 was found, with a reasonable agreement with adjusted (R 2 = 0.9736) value, which indicates that the model has predicted  where A-Oil (%); B-Surfactant (%); and C-Co-surfactant (%) Table 1 lists the effects of independent factors on the globule sizes of various formulations. Negative coefficients of A (oil) and C (co-surfactant) significantly affect the response Y1, showing that the mean globule size was observed to decrease with decrease in the concentration of oil and co-surfactant [36] (see Figure 2). This equation showed A, C, AC, BC, A 2 and C 2 to be significant model terms with p < 0.05. The significance of the model was demonstrated by its model F-value of 66.55 (p < 0.05). An ANOVA evaluation of the quadratic model showed that responses Y1 and Y2 were significantly fitted ( Table 2). The model performed well because the lack of fit's F-value of 0.225 suggested that it was not significant. The quadratic model has a higher R 2 (0.9884) than the other models. An appropriate signal was suggested by the sufficient precession larger than 4 (25.29) [37]. The predicted R 2 and adjusted R 2 values for response Y1 was found, with a reasonable agreement with adjusted (R 2 = 0.9736) value, which indicates that the model has predicted the responses well [38]. When response Y1, or globule size, is given, and A, B, and C have negative coefficients, the average globule size was seen to decrease with an increase in oil, surfactant, and co-surfactant concentrations (see Figure 2). The 3D graph and plots of actual versus predicted particle size are shown in Figure 2.  [38]. When response Y1, or globule size, is given, and A, B, and C ha negative coefficients, the average globule size was seen to decrease with an increase in o surfactant, and co-surfactant concentrations (see Figure 2). The 3D graph and plots of tual versus predicted particle size are shown in Figure 2.     Table 1 lists the influence of independent variables on the PDI of various NEs. The quadratic model was found to be the most appropriate model for the experimental design. The model is suggested to be significant by the model's F-value of 16.42. The large F-value might happen as a result of noise only 0.06% of the time. The lack of fit was indicated by the F-value of 1.13; the lack of fit was not significant in comparison with the pure error [38]. The model was well-fitted since its R 2 value of 0.9548 was greater than that of other (linear, second order) models. Adequate precession of 16.47 that was sufficient (>4) showed that the model was significantly fitted. ANOVA of the quadratic model was calculated and found to be significantly fitted ( Table 2). The 3D graph and plots of actual versus predicted particle PDI are shown in Figure 3.  The %EE of the all NEs formulae were determined and the results are listed in Table 1. The effect of independent variables were studied on %EE. The model is implied to be significant by the model's F-value of 224.63. An F-value this large might happen due to noise only 0.01% of the time. Model terms are considered significant when the p-value is lower than 0.0500. In this case, the model terms A, C, AC, BC, A 2 , B 2 , and C 2 were significant. Model terms are not significant if the value is higher than 0.1000. Model reduction may The %EE of the all NEs formulae were determined and the results are listed in Table 1. The effect of independent variables were studied on %EE. The model is implied to be Gels 2023, 9, 95 7 of 20 significant by the model's F-value of 224.63. An F-value this large might happen due to noise only 0.01% of the time. Model terms are considered significant when the p-value is lower than 0.0500. In this case, the model terms A, C, AC, BC, A 2 , B 2 , and C 2 were significant. Model terms are not significant if the value is higher than 0.1000. Model reduction may enhance your model if it has a large number of useless terms. The F-value for the lack of fit, which is 1.31, indicates that the lack of fit is not significant in comparison with pure error. A large lack of fit F-value has a 38.73% chance of being caused by noise. We want the model to fit; thus, a non-significant lack of fit is ideal ( Table 2). The 3D graph and plots of actual versus predicted particle %EE are shown in Figure 4.

Measurement of Globule Size, PDI, %EE and Zeta Potential
The prepared DSM-loaded NEs were found to have a mean globule size in the r of 34-65 nm and PDI 0.055-0.128. The ideal mean globule size for topical distribut below 200 nm, with a PDI below 0.3 [39]. This is because the globules have a large su area that is suitable for quick pore transport and good for topical gel. The optimized D loaded NEs were determined as globule size (41 nm), PDI (0.073) and ZP (−23.4 mV) optimized DSM-loaded NEs were further incorporated into carbopol gel. The %EE o pared DSM-loaded NEs (F1-F17) were found to be in the range of 64-94% (Table 1)

Measurement of Globule Size, PDI, %EE and Zeta Potential
The prepared DSM-loaded NEs were found to have a mean globule size in the range of 34-65 nm and PDI 0.055-0.128. The ideal mean globule size for topical distribution is below 200 nm, with a PDI below 0.3 [39]. This is because the globules have a large surface area that is suitable for quick pore transport and good for topical gel. The optimized DSM-loaded NEs were determined as globule size (41 nm), PDI (0.073) and ZP (−23.4 mV). This optimized DSM-loaded NEs were further incorporated into carbopol gel. The %EE of prepared DSM-loaded NEs (F1-F17) were found to be in the range of 64-94% (Table 1). Based on the results, DSM-loaded NEs (F1) were optimized with globule size (41 nm), PDI (0.073), ZP (−23.4 mV) ( Figure 5) and %EE (87%).

Determination of Viscosity, Refractive Index (RI) and pH
The viscosity, RI and pH of the optimized DSM-loaded NEs (F1) was measured as 77.1 ± 2.42 cP, 1.28 ± 0.03, and 4.6 ± 0.08, respectively. The ideal RI value, which demonstrates the transparency and isotropic quality of NEs formulation, was measured. It was determined that the pH was 4.6 ± 0.08, which is completely compatible with the pH of Gels 2023, 9,95 8 of 20 the skin and would not irritate the skin. It was found that the optimized DSM-loaded NEs (F1) have a 98.59 ± 0.03% transmittance, which is closest to 100% transmittance, indicating formulation transparency and clarity [40,41].

Measurement of Globule Size, PDI, %EE and Zeta Potential
The prepared DSM-loaded NEs were found to have a mean globule size in the range of 34-65 nm and PDI 0.055-0.128. The ideal mean globule size for topical distribution is below 200 nm, with a PDI below 0.3 [39]. This is because the globules have a large surface area that is suitable for quick pore transport and good for topical gel. The optimized DSMloaded NEs were determined as globule size (41 nm), PDI (0.073) and ZP (−23.4 mV). This optimized DSM-loaded NEs were further incorporated into carbopol gel. The %EE of prepared DSM-loaded NEs (F1-F17) were found to be in the range of 64-94% (Table 1)

Thermodynamic Stability Studies
To check the potential of metastable or unstable NEs, thermodynamic stability was explored. After centrifugation, heating and cooling, and freeze-thaw cycles, it was found that the optimized DSM-loaded NEs (F1) were thermodynamically stable. Results of thermodynamic stability shows that the DSM-loaded NEs (F1) had a long shelf-life. The optimized DSM-loaded NEs (F1) were determined to be thermodynamically stable [42] and found to be suitable for incorporation into gel.

Transmission Electron Microscopy (TEM)
Using TEM, the globules of optimized DSM-loaded NEs (F1) were morphologically characterized. The TEM picture ( Figure 6) shows a spherical shape and an average size of about 40 to 90 nm. The globules are evenly distributed. The majority of the particles in the TEM pictures measure less than 100 nm in size. Gels 2023, 9, x FOR PEER REVIEW 9 of 21

Determination of Viscosity, Refractive Index (RI) and pH
The viscosity, RI and pH of the optimized DSM-loaded NEs (F1) was measured as 77.1 ± 2.42 cP, 1.28 ± 0.03, and 4.6 ± 0.08, respectively. The ideal RI value, which demonstrates the transparency and isotropic quality of NEs formulation, was measured. It was determined that the pH was 4.6 ± 0.08, which is completely compatible with the pH of the skin and would not irritate the skin. It was found that the optimized DSM-loaded NEs (F1) have a 98.59 ± 0.03% transmittance, which is closest to 100% transmittance, indicating formulation transparency and clarity [40,41].

Thermodynamic Stability Studies
To check the potential of metastable or unstable NEs, thermodynamic stability was explored. After centrifugation, heating and cooling, and freeze-thaw cycles, it was found that the optimized DSM-loaded NEs (F1) were thermodynamically stable. Results of thermodynamic stability shows that the DSM-loaded NEs (F1) had a long shelf-life. The optimized DSM-loaded NEs (F1) were determined to be thermodynamically stable [42] and found to be suitable for incorporation into gel.

Transmission Electron Microscopy (TEM)
Using TEM, the globules of optimized DSM-loaded NEs (F1) were morphologically characterized. The TEM picture ( Figure 6) shows a spherical shape and an average size of about 40 to 90 nm. The globules are evenly distributed. The majority of the particles in the TEM pictures measure less than 100 nm in size. The F1-loaded gel was assessed for its pH and drug content. The gels had a pH of 6.1 ± 0.3, which is ideal for skin application. DSM content in F1-loaded gel was measured as 98 ± 4.1%. With a uniform distribution of DSM, the maximum amount of medication in  The F1-loaded gel was assessed for its pH and drug content. The gels had a pH of 6.1 ± 0.3, which is ideal for skin application. DSM content in F1-loaded gel was measured as 98 ± 4.1%. With a uniform distribution of DSM, the maximum amount of medication in the gel can be determined from the results.

Spreadability
Excellent spreading is required to evenly cover the wound with the drug for a quicker wound-healing process [43]. The spreadability value, which was indicated in the area, is a crucial factor in how evenly the gel will spread over the skin. The area, which was determined to have a decent spreading capacity, was 6.13 ± 0.19 cm 2 .

In Vitro Release Studies and Kinetics
A comparative in vitro release profile of pure DSM suspension, optimized DSM-loaded NEs (F1) and F1-loaded gel were presented in Figure 7. The carbopol gel system's release behavior indicated a dual-release pattern; the early stages of drug release appeared to be a quick release in the first 2 h, while the later stages were a sustained release for a longer period of time (24 h). The amount of DSM released from pure DSM suspension, optimized DSM-loaded NEs (F1) and F1-loaded gel in 24 h was 28.76%, 63.32% and 70.54%, respectively. Due to its poor aqueous solubility, pure DSM demonstrated a poor release. The release was observed to be significantly increased (p < 0.05) following the encapsulation of DSM into NEs. Lower globule size of optimized DSM-loaded NEs (F1) is possible due to presence of Lauroglycol-90, tween-80 and THP as oil, surfactant and co-surfactant, respectively. Due to the larger surface area, smaller droplets would have a higher drugrelease profile [41]. The release kinetics equations were used to study the DSM release pattern from optimized DSM-loaded NEs (F1) and F1-loaded gels. Applying the kinetic models, it was found that Korsmeyer-Peppas model could adequately describe the release of DSM from both NEs and gels. The Korsmeyer-Peppas model had the highest coefficient of correlation values among the applied release models (R 2 = 0.9890 and 0.9793 for DSMloaded NE (F1) and F1-gel, respectively, as shown in Table 3). The release exponents were also calculated in light of the values of slopes R 2 obtained for the Korsmeyer-Peppas model, and the results suggested that the mechanism of drug release from DSM-loaded NE (F1) and F1-gel were of the Fickian diffusion type (Table 3) [44]. Excellent spreading is required to evenly cover the wound with the drug for a quicker wound-healing process [43]. The spreadability value, which was indicated in the area, is a crucial factor in how evenly the gel will spread over the skin. The area, which was determined to have a decent spreading capacity, was 6.13 ± 0.19 cm 2 .

In Vitro Release Studies and Kinetics
A comparative in vitro release profile of pure DSM suspension, optimized DSMloaded NEs (F1) and F1-loaded gel were presented in Figure 7. The carbopol gel system's release behavior indicated a dual-release pattern; the early stages of drug release appeared to be a quick release in the first 2 h, while the later stages were a sustained release for a longer period of time (24 h). The amount of DSM released from pure DSM suspension, optimized DSM-loaded NEs (F1) and F1-loaded gel in 24 h was 28.76%, 63.32% and 70.54%, respectively. Due to its poor aqueous solubility, pure DSM demonstrated a poor release. The release was observed to be significantly increased (p < 0.05) following the encapsulation of DSM into NEs. Lower globule size of optimized DSM-loaded NEs (F1) is possible due to presence of Lauroglycol-90, tween-80 and THP as oil, surfactant and cosurfactant, respectively. Due to the larger surface area, smaller droplets would have a higher drug-release profile [41]. The release kinetics equations were used to study the DSM release pattern from optimized DSM-loaded NEs (F1) and F1-loaded gels. Applying the kinetic models, it was found that Korsmeyer-Peppas model could adequately describe the release of DSM from both NEs and gels. The Korsmeyer-Peppas model had the highest coefficient of correlation values among the applied release models (R 2 = 0.9890 and 0.9793 for DSM-loaded NE (F1) and F1-gel, respectively, as shown in Table 3). The release exponents were also calculated in light of the values of slopes R 2 obtained for the Korsmeyer-Peppas model, and the results suggested that the mechanism of drug release from DSM-loaded NE (F1) and F1-gel were of the Fickian diffusion type (Table 3)

Acute Dermal Toxicity
The acute dermal toxicity test confirmed the safety of F1-loaded gel application. After 24 h of treatment, the shaved region showed no signs of erythema or edema. When rats were monitored for 14 days after the gel application, neither mortality nor any serious modifications in their behavior or respiratory patterns, any inability to consume food or water, or abnormal postural alignment were found, which bore close resemblance to our previous study [24].

Excision Wound Model
In comparison with the negative control group, topical application of 2% F1-loaded gel demonstrated a substantial impact on the wound-healing process. Figure 8 depicts the progressive wound contraction induced by the use of base gel, 2% F1-loaded gel, and 2% fusidic acid gel. The 2% F1-loaded and fusidic acid gel facilitates wound contraction significantly from the 7th day to 21st day compared to NC ( Figure 9).

Acute Dermal Toxicity
The acute dermal toxicity test confirmed the safety of F1-loaded gel application. After 24 h of treatment, the shaved region showed no signs of erythema or edema. When rats were monitored for 14 days after the gel application, neither mortality nor any serious modifications in their behavior or respiratory patterns, any inability to consume food or water, or abnormal postural alignment were found, which bore close resemblance to our previous study [24].

Excision Wound Model
In comparison with the negative control group, topical application of 2% F1-loaded gel demonstrated a substantial impact on the wound-healing process. Figure 8 depicts the progressive wound contraction induced by the use of base gel, 2% F1-loaded gel, and 2% fusidic acid gel. The 2% F1-loaded and fusidic acid gel facilitates wound contraction significantly from the 7th day to 21st day compared to NC ( Figure 9).

Microscopic Evaluation of the Wound
The photomicrograph of the NC group stained with H&E shows severe tissue age in the form of necrosis (N) due to the absence of nuclei, degeneration (D) in th of vacuoles, as well as occlusion of blood vessel (O) through a large spot of hyaline rial. Moreover, the blood vessel walls suffer from injury, leading to hemorrhag Fusidic acid gel treatment shows an improved and almost normal tissue sample b suffering from toxic effect in the form of degeneration (D) and necrosis (N). Howev loaded applied on a wound shows a much improved and almost normal tissue sam The collagen fiber loss is visible on the photomicrograph of the NC group usi MT stain as a light blue tint (example is indicated by yellow arrow). Fusidic acid-t skin tissues exhibit increased collagen fiber condition as a dark blue color. Treatmen F1-loaded gel shows almost normal and significantly improved collagen fiber con ( Figure 10).
The photomicrograph of the NC group stained with the Verhoeff method sho loss of elastic fibers as a lower density of black color (example is indicated by yell row). In comparison with the NC group, the photomicrograph of the positive c group after treatment with fusidic acid gel and F1-loaded gel reveals an improvem the condition of the elastic fibers ( Figure 10).

Microscopic Evaluation of the Wound
The photomicrograph of the NC group stained with H&E shows severe tissue damage in the form of necrosis (N) due to the absence of nuclei, degeneration (D) in the form of vacuoles, as well as occlusion of blood vessel (O) through a large spot of hyaline material. Moreover, the blood vessel walls suffer from injury, leading to hemorrhage (H). Fusidic acid gel treatment shows an improved and almost normal tissue sample but still suffering from toxic effect in the form of degeneration (D) and necrosis (N). However, F1-loaded applied on a wound shows a much improved and almost normal tissue sample.
The collagen fiber loss is visible on the photomicrograph of the NC group using the MT stain as a light blue tint (example is indicated by yellow arrow). Fusidic acid-treated skin tissues exhibit increased collagen fiber condition as a dark blue color. Treatment with F1-loaded gel shows almost normal and significantly improved collagen fiber condition ( Figure 10).

Microscopic Evaluation of the Wound
The photomicrograph of the NC group stained with H&E shows severe tissue da age in the form of necrosis (N) due to the absence of nuclei, degeneration (D) in the fo of vacuoles, as well as occlusion of blood vessel (O) through a large spot of hyaline ma rial. Moreover, the blood vessel walls suffer from injury, leading to hemorrhage ( Fusidic acid gel treatment shows an improved and almost normal tissue sample but s suffering from toxic effect in the form of degeneration (D) and necrosis (N). However, loaded applied on a wound shows a much improved and almost normal tissue sampl The collagen fiber loss is visible on the photomicrograph of the NC group using MT stain as a light blue tint (example is indicated by yellow arrow). Fusidic acid-trea skin tissues exhibit increased collagen fiber condition as a dark blue color. Treatment w F1-loaded gel shows almost normal and significantly improved collagen fiber condit ( Figure 10).
The photomicrograph of the NC group stained with the Verhoeff method shows loss of elastic fibers as a lower density of black color (example is indicated by yellow row). In comparison with the NC group, the photomicrograph of the positive con group after treatment with fusidic acid gel and F1-loaded gel reveals an improvemen the condition of the elastic fibers ( Figure 10). The photomicrograph of the NC group stained with the Verhoeff method shows the loss of elastic fibers as a lower density of black color (example is indicated by yellow arrow). In comparison with the NC group, the photomicrograph of the positive control group after Gels 2023, 9,95 12 of 20 treatment with fusidic acid gel and F1-loaded gel reveals an improvement in the condition of the elastic fibers ( Figure 10).

Evaluation of Anti-Inflammatory
In Table 4, the percentages of inhibition of edema by F1-loaded gel and fusidic acid gels were shown as 42.51% and 50.69%, respectively, compared to the control. Based on these findings, it can be said that the F1-loaded gel produced an anti-inflammatory effect that was at least 1.2 times stronger than that of the fusidic gel. The F1-loaded nanogel provides highest concentration of DSM at the target side and because the drug can penetrate the skin more easily due to its nanosize, the nanogel formulation may have the strongest anti-inflammatory effects [45,46]. Values are expressed as mean ± S.E.M., n = 6 rats/group, symbol *** indicates significance compared to NC group at p < 0.05.

Conclusions
In this study, the solubility of diosmin in numerous oils, surfactants and co-surfactants was compared and the best oil, surfactant and co-surfactant blend was selected for the development of NEs. The NE was prepared through an aqueous titration method using QBD design. NE with uniform globules size, PDI and higher EE was selected for the incorporation into carbopol 940 gel. All the pharmaceuticals characterizations were considered and, after showing the optimum and fickian drug release, optimized DSM-NEs (F1) loaded gel was further tested in vivo for anti-inflammatory and wound-healing activity. The excision model wounds were examined histopathologically. On the basis of gross necropsis and histological images from the rat wound, it can be concluded that there was a faster wound-healing process observed in the test animals compared to the STD and control. Finally, in vitro and in vivo results revealed that diosmin-encapsulated nanoemulsionloaded carbopol 940 gels would be a promising topical formulation for wound-healing and anti-inflammatory effects with minimal/acceptable acute dermal toxicity.

Solubility Studies
To choose the best oil phase for the development of the formulation, solubility experiments were conducted. Numerous oils, including LG-90 ® , PGMC, kollidon-EL (K-EL) olive oil (OO), and sesame oil (SO) were used to test the solubility of DSM. The same experiments were performed with surfactants (tween 20, and tween 80) and co-surfactants (ethyl alcohol and Transcutol HP) [24]. Briefly, an excess amount of DSM in 2 mL of each of the selected oils, surfactants, and co-surfactants was dissolved and mixed, followed by shaking on a biological shaker (LBS-030S, Lab Tech, Jeju, Korea) for 72 h days at 37 ± 1 • C, 100 rpm. The DSM concentration in each component was measured in triplicate using HPLC at 346 nm [47].

Experimental Design
The development of DSM-loaded NEs was carried out utilizing a three-level Box-Behnken experimental design (BBD) with three independent factors using Design Expert software (version 13.0 Stat-Ease, Inc., Minneapolis, MN, USA). The Box-Behnken design model for response surface approach is useful because it enables: (i) evaluation of the quadratic model parameters; (ii) creation of following designs; (iii) identification of model's lack of fit; (iv) utilization of blocks; (v) the avoidance of severe experimenting because it could lead to disappointing results. In addition, it performs fewer runs of experiments with three factors. The concentration of oil (LG ® 90; X1), concentration of surfactant (tween 80; X2), and concentration of co-surfactant (THP; X3) were the three parameters chosen for this investigation. They were grouped into three levels and assigned the codes +1, 0, and −1 for high, moderate, and low values, respectively. The oils, surfactants, and co-surfactants were selected based on solubility study. The following independent variables or components were chosen: (A) oil concentration (10-40% w/w), (B) surfactant concentration (10-30% w/w), and (C) co-surfactant concentration (10-30% w/w). The mean globule size (Y1), PDI (Y2) and entrapment efficiency (Y3) were selected as the dependent variables/responses in order to optimize the NEs. The software created a total of 17 tests, and the experiments were carried out in a random order. The independently coded variables are displayed in Table 1. Formulations were prepared and further examined for the 17 batches that were produced.

Selection of Optimized Formulation
The software's point prediction, which explained the formulation's center point, was used to select the optimum formulation. The composition of optimized NEs had oil (LG90, 25% w/w), surfactant (tween, 20% w/w) and co-surfactant (THP, 20% w/w), the predicted values of globule size (40.2 nm), PDI (0.0798) and %EE (87.8%). However, the experimental values of optimized NEs were determined to be close to the predicted values of globule size (41 ± 0.07 nm), PDI (0.073 ± 0.008) and %EE (87 ± 0.81%). This optimized formulation was further evaluated and incorporated into gel.

Preparation of DSM-Loaded NEs
The spontaneous emulsification method was used to develop NEs. Briefly, 10 mg of DSM were precisely weighed and added to the oil phase (LG-90 ® ). The mixture was then added, together with surfactant (Tween 80) and co-surfactant (THP), and vortexed for 10 min at 250 rpm. The produced mixture was then gradually added to the aqueous phase that had been magnetically agitated. The resulting transparent NEs were further evaluated for globule size, PDI, zeta potential and %EE.

Measurement of Globule Size, PDI, %EE and Zeta Potential
All developed DSM-loaded NEs formulations (F1-F17) were assessed for their globule size and PDI using the dynamic light scattering (DLS) method with a Zetasizer Nano ZS device (Malvern Instruments, Worcestershire, UK). The degree of distribution and monodispersity of the globules was determined using the polydispersity index (PDI), and the PDI with <0.3 value was regarded as monodisperse [48]. Zeta potential measurements provide an indicator of the strength of repulsion and attraction between NEs globules as these detect the electric charges on the surface of globules [49]. A Malvern Zetasizer (Malvern Instruments, Worcestershire, UK) was used to measure the zeta potential of each formulation at a temperature of 25 ± 1 • C. Freshly prepared samples were diluted (1:500), put into an electrode cuvette, and their NSPs, PDI, and ZP values were assessed. The EE of DSM-loaded NEs formulations (F1-F17) were determined. The DSM-loaded NEs (0.5 mL) were appropriately diluted with methanol, centrifuged at 6000 rpm for 10 min and analyzed for content using HPLC at 346 nm [47]. The %EE of the DSM-loaded NEs formulations were estimated using the following equation: Diosmin analyzed in NEs Diosmin added NEs × 100

Determination of Refractive Index (RI) and pH
With the help of a Brook-field viscometer, DV II ultra, RV cone and plate (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA), the viscosity of the optimized DSMloaded NEs (F1) was assessed. The Abbe's refractometer (Precision Testing Instruments Laboratory, Germany) was used to check the RI values of developed optimized DSM-loaded NEs (F1) at a temperature of 25 ± 2 • C. The optimized NEs had their pH and %T measured at 550 nm using a UV/Vis spectrophotometer (Jasco-UV-visible spectrophotometer, Model: V-630, Tokyo, Japan).

Thermodynamic Stability Studies
The developed optimized DSM-loaded NEs (F1) was examined for different thermodynamic stability studies in order to check the stability. According to the published protocol, tests on optimized DSM-loaded NEs (F1) included centrifugation, heating and cooling (three cycles between 45 • C and 25 • C), and freeze-pump-thaw cycles (three cycles between −25 • C and 25 • C) [42,50]. The visual analysis was used to evaluate the thermodynamic stability tests for phase separation, precipitation, and coalescence.

Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) was used to examine the size and shape of the optimized DSM-loaded NEs (F1) using a Jeol TEM model JEM-1101 (Tokyo, Japan). The TEM analysis provides the insight of NE morphology.

Preparation of DSM-Loaded NEs Gels
The carbopol 940 (1%, w/v) gelling agent was used to successfully incorporate the optimized DSM-loaded NEs (2%, w/v) into the carbopol gel. A few drops (2-3 drops) of triethanolamine (0.5%) were added to carbopol 940 dispersion to aid in the polymer's soaking and swelling; the mixture was then kept in the fridge overnight, and, after 24 h, the optimized DSM-loaded NEs (F1) was mixed with carbopol 940 (1%, w/v) gel and methylparaben (0.1%) using magnetic stirring at a speed of 250 rpm [51]. The developed gels was further evaluated (Figure 11). Gels 2023, 9, x FOR PEER REVIEW 15 of 21 of DSM-loaded NEs formulations (F1-F17) were determined. The DSM-loaded NEs (0.5 mL) were appropriately diluted with methanol, centrifuged at 6000 rpm for 10 min and analyzed for content using HPLC at 346 nm [47]. The %EE of the DSM-loaded NEs formulations were estimated using the following equation:

Determination of Refractive Index (RI) and pH
With the help of a Brook-field viscometer, DV II ultra, RV cone and plate (Brookfield Engineering Laboratories, Inc., Middleboro, MA, USA), the viscosity of the optimized DSM-loaded NEs (F1) was assessed. The Abbe's refractometer (Precision Testing Instruments Laboratory, Germany) was used to check the RI values of developed optimized DSM-loaded NEs (F1) at a temperature of 25 ± 2 °C. The optimized NEs had their pH and %T measured at 550 nm using a UV/Vis spectrophotometer (Jasco-UV-visible spectrophotometer, Model: V-630, Tokyo, Japan).

Thermodynamic Stability Studies
The developed optimized DSM-loaded NEs (F1) was examined for different thermodynamic stability studies in order to check the stability. According to the published protocol, tests on optimized DSM-loaded NEs (F1) included centrifugation, heating and cooling (three cycles between 45 °C and 25 °C), and freeze-pump-thaw cycles (three cycles between −25 °C and 25 °C) [42,50]. The visual analysis was used to evaluate the thermodynamic stability tests for phase separation, precipitation, and coalescence.

Transmission Electron Microscopy (TEM)
Transmission electron microscopy (TEM) was used to examine the size and shape of the optimized DSM-loaded NEs (F1) using a Jeol TEM model JEM-1101 (Tokyo, Japan). The TEM analysis provides the insight of NE morphology.

Preparation of DSM-Loaded NEs Gels
The carbopol 940 (1%, w/v) gelling agent was used to successfully incorporate the optimized DSM-loaded NEs (2%, w/v) into the carbopol gel. A few drops (2-3 drops) of triethanolamine (0.5%) were added to carbopol 940 dispersion to aid in the polymer's soaking and swelling; the mixture was then kept in the fridge overnight, and, after 24 h, the optimized DSM-loaded NEs (F1) was mixed with carbopol 940 (1%, w/v) gel and methylparaben (0.1%) using magnetic stirring at a speed of 250 rpm [51]. The developed gels was further evaluated (Figure 11).

pH and Drug Content
The F1-loaded gel, weighing about 1 g, was dissolved in milli-Q water (100 mL). The pH of the gel formulation was determined by dipping an electrode of pH meter (Adwa, Model-AD1000, Szeged, Hungary), which had been previously calibrated with standard buffers (pH 4, 7, and 9). The pH values were measured as the mean of three readings with standard deviations (Mean ± SD, n = 3). The content of DSM in the F1-loaded gel was measured by dissolving 1 g of gel in methanol (10 mL). The sample was then diluted, sonicated and analyzed for DSM content in the gel using the HPLC method.

Spreadability
Briefly, placing the F1-loaded gel on a clean petri plate and noting its initial diameter, a second petri plate was placed on top of the first, and a weight (100 g) was placed over the second petri plate for 30 s to test the spreadability of the gel. The final dimeter was measured, and the spreadability percentage was computed using the given equation: (1)

In Vitro Diffusion Studies and Release Kinetics
To conduct in vitro release tests of the pure DSM solution, optimized DSM-loaded NEs (F1), and F1-loaded gel, Franz diffusion cells with a 10 mL capacity were used. As a diffusion membrane, the dialysis membrane (Avg. Mol wt. 14000 Dalton, Himedia, India) was soaked in phosphate buffer (PB), with a pH of 7.4, for 24 h before the experiment. The diffusion cell was filled with phosphate buffer and a dialysis membrane was attached to the cell. The assembly was kept at 37 ± 0.5 • C using a thermostatically controlled, timeprogrammed magnetic stirrer at 100 rpm. The samples (0.5 mL) were taken out at regular time intervals of 24 h (0, 0.5, 1, 2, 3, 6, 12 and 24 h) and replaced with an equal volume of fresh PB (pH 7.4) to maintain the sink condition. The samples were then evaluated for drug content using HPLC at 346 nm [47]. The released amount of drug was then plotted against time, and fitted to the mathematical models to obtain the release mechanism. The following model equations were used to treat release data [52]: where K is the kinetic rate constant, Mt/M 0 is the fraction of DSM released at time t, and n is the diffusion exponent, the value of which describes the release mechanism, wherein if n = 0.5 (Fickian diffusion), 0.5 < n < 1.0 (Anomalous non-Fickian transport), and for n = 1.0 (Case-II, relaxational).

Experimental Animals
Healthy, Wistar albino rats (180-200 gm, 3-4 months of age) of both sexes were received from the College of Pharmacy's Animal Care Department at the Prince Sattam Bin Abdulaziz University (PSAU), Saudi Arabia. The rats were housed in clean cages under standard laboratory conditions with a 12/12 h light/dark cycle, a temperature of 22 ± 2 • C, with an access to a pellet diet and water ad libitum. Before the experiment began, all animals were given a week to get used to the lab environment. The animal study was approved by the ethical committee of the Prince Sattam bin Abdulaziz University (SCBR-030-2022), and conducted according to the science and ethical principles for animal care, at the college of pharmacy, PSAU, Saudi Arabia.

Acute Dermal Toxicity
Acute dermal toxicity testing was carried out in accordance with OECD recommendation number 434. [53]. We used a total of 10 female Wistar rats (8-12 weeks). The dorsal portion of the trunk of animals with normal skin texture had about 10% of its body surface area removed 24 h before the investigation. They were divided into two groups (treatment and control). DSM-loaded gel was applied evenly to the test site, wrapped with a porous gauze bandage, and left there for a 24-h exposure period. By applying the 2% gel, a sighting study was first conducted to ascertain the starting dose. At 60 min, 24 h, 48 h, and 72 h after the initial examination, all animals were scored for the presence of erythema and edema. There were no deaths or skin rashes within 24 h, and four more rats from each group were utilized, and the same dose of gel was given. The rats were monitored daily, at least once during the first 30 min, frequently during the first 24 h, and with special attention given during the first 4 h, unless they were found dead, or any adverse dermal reaction appeared right after dosing.

Excision Wound Model
The excision model was conducted on three groups of six rats each. Animals in group I (NC) were treated as negative control and given topically applied 2% base gel. Groups II (the treated group) and III (the positive control group) received, respectively, 2% w/w DSM-loaded NE (F1) gel and fusidic acid (FA) gel.
The rats were anesthetized with chloral hydrate (400 mg/kg, IP). The animals' dorsal furs were machine-shaved before the wound area was prepared. Then, their dorsothoracic region's fur was cut off. Permanent marker was used to create a 200 mm 2 circular mark, which was then completely excised to create a wound using sterile forceps and scissors. This was considered day 0. For the examination of wound-healing activity, the rats were given base, DSM-loaded (F1) gel, and fusidic acid gel starting on day one. The test groups' wounds were treated with all of the preparations daily until they had fully healed. Every seven days, the wound area was measured using a transparent sheet and permanent pen. On graph paper with a 1 mm 2 scale, the transparent sheet was laid down and sketched out. The wound-healing activities were assessed according to the period of epithelialization and the percentage of wound contraction [54,55]. The percentage of wound contraction was calculated as follows: % Wound Contraction = Wound Area on day 0 − Wound Area on day n Wound Area on day 0 × 100 (6) where n is the number of measurement-taking days.

Microscopic Evaluation of the Wound
Skin tissue samples were collected from all the groups into suitable bottles. Immediately after collection, these samples were immersed in a solution of 10% formalin for fixation. Then, tissue processing was accomplished using an automatic machine for tissue processing (ASP300s, Leica Biosystems, Deer Park, IL, USA). Then, using a rotary microtome, blocks of paraffin wax were used to implant tissue samples and cut these into thin sections of 5 µm thickness (SHUR/Cut 4500, TBS, Durham, NC, USA) [50]. Three sections of each tissue block were taken for staining. One section was stained using the common staining procedure of hematoxylin and eosin (H&E). The second section was stained using the Masson tricrome (MT) special stain method for connective tissue fibers; and the third specimen was stained using the Verhoeff special stain method [50,51]. After proper staining, a histopathologist evaluated photomicrographs for pathologies and examined wound changes under a light microscope.

In Vivo Anti-Inflammatory Activity
The DSM-loaded NE (F1) gel's anti-inflammatory efficacy was tested using a rat model of carrageenan-induced hind paw edema [56][57][58]. Rats were chosen for each group so that the groups' average body weights were as similar as possible. The animals that had been fasted for the previous night (n = 18) were grouped into three groups of six rats each to assess the anti-inflammatory activity. The plantar surface of the left hind paw was topically applied to the rats in group 1 (NC) by gently rubbing the 1% base gel 50 times with the index finger. F1-loaded gels and fusidic acid gel were given to Group 2 (the treated group) and Group 3 (the positive control group), respectively, topically on the plantar surface of the left hind paw with the same mode of application as the base gel. After 3 h since dosing, 0.1 mL of a 1% carrageenan solution (in normal saline) was injected subplantarly into the treated paw. Using a plethysmometer, the paw edema was measured immediately (0 h) and again 3 h after the carrageenan injection. The following equation was used to determine the percentage of the paw's swelling: % Paw Edema Inhibition = % Swelling of control group − % Swelling of treated group % Swelling of control group × 100 (8) where Vi is the initial paw volume and V is the paw volume 3 h after the carrageenan injection. The average amount of paw edema in the drug-treated group was compared to that in the control group, and the percentage inhibition of edema caused by the treatment was computed using the formula below.

Statistical Analysis
The mean and standard error of the mean (SEM) were used to express all the results. One-way analysis of variance (ANOVA), followed by the Bonferroni test, was used to statistically analyze the results. The results were deemed statistically significant at a p-value of <0.05 at 95% confidence interval (CI). All data processing was carried out using the GraphPad program (Graph Pad, San Diego, CA, USA).  Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.