Development of Apremilast Nanoemulsion-Loaded Chitosan Gels: In Vitro Evaluations and Anti-Inflammatory and Wound Healing Studies on a Rat Model

Apremilast (APL) has profound anti-inflammatory and wound healing activity, alongside other dermal care. This study aims to develop APL-loaded NEs (ANE1-ANE5) using eucalyptus oil (EO) as the oil and Tween-80 and transcutol-HP (THP) as a surfactant and co-surfactant, respectively. The prepared NEs were then evaluated based on mean droplet size (12.63 ± 1.2 nm), PDI (0.269 ± 0.012), ZP (−23.00 ± 5.86), RI (1.315 ± 0.02), and %T (99.89 ± 0.38) and ANE4 was optimized. Further, optimized NEs (ANE4) were incorporated into chitosan gel (2%, w/v). The developed ANE4-loaded chitosan gel was then evaluated for pH, spreadability, in vitro diffusion, and wound healing and anti-inflammatory studies. Moreover, in vivo studies denoted improved anti-inflammatory and wound healing activity and represented a decrease in wound size percentage (99.68 ± 0.345%) for the APNE2 gel test compared to a negative control (86.48 ± 0.87%) and standard control (92.82 ± 0.34%). Thus, the formulation of ANE4-loaded chitosan gels is an efficient topical treatment strategy for inflammatory and wound healing conditions.


Introduction
Apremilast (APL) is a selective PDE4 inhibitor indicated for plaque psoriasis and psoriatic arthritis [1]. Its recommended dosage regimen is 10 mg as a starting dose twice daily followed by 30 mg in five days and 30 mg taken thereafter. APL is considered a small molecule with a molecular weight of 460.50 with a half-life of 6-9 h. It is a pale yellow powder that is insoluble in water and has a BCS-IV classification [2]. APL acts as a phosphodiesterase 4 (PDE4) inhibitor that suppresses the cyclic adenosine monophosphate (cAMP) degradation to AMP in cells. The increased intracellular secondary messenger cAMP, due to the activation of adenylyl cyclase, influences a wide array of cellular functions; activates transcription factor nuclear factor-kappa B (NF-κB) and the expression of proinflammatory cytokines such as IL-23, tumor necrosis factor-α (TNF-α), and interferongamma (IFN-γ); and regulates anti-inflammatory interleukins [3,4]. Briefly, APL increases intracellular cAMP levels and then modulates the production of inflammatory mediators indirectly [5]. Inflammation may also be instigated by the derivative of arachidonic acid and

Pseudo-Ternary Phase Diagram
The pseudo-ternary phase diagrams were constructed using EO (oil), Smix (Tween-80 and THP), and water ( Figure 2). When Tween-80 was utilized without THP (Smix ratio 1:0), it was observed that Tween-80 alone is insufficient to generate stable NEs. The zone of NEs area in the phase diagram grew immensely when surfactants and co-surfactants were used in equal proportions (Smix ratio 1:1) compared to Smix ratio 1:0 [23,24]. THP reduces the interfacial tension and makes the interfacial film flexible enough to take on the various curvatures necessary to create NEs over a wide variety of compositions. The zone of NEs area was increased when the content of THP as a co-surfactant was increased (Smix ratio 1:2) ( Figure 2). The increase in the NEs area is possibly due to a high concentration of co-surfactant, which increases the solubilization capacity of NEs.

Pseudo-Ternary Phase Diagram
The pseudo-ternary phase diagrams were constructed using EO (oil), Smix (Tween-80 and THP), and water ( Figure 2). When Tween-80 was utilized without THP (Smix ratio 1:0), it was observed that Tween-80 alone is insufficient to generate stable NEs. The zone of NEs area in the phase diagram grew immensely when surfactants and co-surfactants were used in equal proportions (Smix ratio 1:1) compared to Smix ratio 1:0 [23,24]. THP reduces the interfacial tension and makes the interfacial film flexible enough to take on the various curvatures necessary to create NEs over a wide variety of compositions. The zone of NEs area was increased when the content of THP as a co-surfactant was increased (Smix ratio 1:2) ( Figure 2). The increase in the NEs area is possibly due to a high concentration of co-surfactant, which increases the solubilization capacity of NEs.

Thermodynamic Stability Study
Thermodynamic stability was accessed to exclude the possibility of metastable/unstable NEs. All developed APL-loaded NEs (ANE1-ANE5) were observed thermodynamically stable after centrifugation, heating and cooling and freeze-thaw cycles. Results of thermodynamic stability reflects long shelf life to the ANE4, relatively as compared to other NEs. Among all developed APL-loaded NEs (ANE1-ANE5), ANE4 was found thermodynamically stable. Therefore, based on mean droplet size, PDI, ZP, RI, %T and thermal stability data, ANE4 was found optimum and further subjected for incorporation into chitosan gel.

TEM Images
The optimized NEs (ANE4), microphotograph represents spherical shape with rough surface and size was found in the nano-range. The rough surface of the nanoemulsion might be due to sample preparation during TEM studies ( Figure 4). The size can be correlated with the results of globule size showed by photon correlation spectroscopy provided by Zetasizer.

Evaluation of APL-Loaded NEs Chitosan Based Gel
The pH measurement govern skin compatibility with developed gel, suitable for topical application and free from allergic reaction and skin irritation. The pH values of ANE4loaded gel was noted to be 6.8 ± 1.2, which is optimum for dermal application. The success of topical gel depends on the spreadability, it's an important parameter on which therapeutic efficacy relies. A small shear is required for uniform spreadability of gels to the skin [28]. Spreadability of the developed NE4-loaded gels exhibited the optimal spreadability over the plexy glass plates the area of formulation spread was observed as be 8.6 cm, suggesting practically excellent spreadability.

Diffusion Study and Release Kinetics Models
Comparative in-vitro diffusion study was performed for pure drug (APL), optimized NEs (NE4) and ANE4-loased gel ( Figure 6). Cumulative percentage drug release was found 18.79% from pure APL suspension, 54.88% and 84.17% from optimized NEs (ANE4) and ANE4-loaded gel after 24 h, respectively. An improved drug release was observed from developed topical gel due to its nanosize carrier, which could facilitate drug penetration across the dermal layer and improve therapeutic concentration at the local site [29]. The release kinetics was also determined by fitting the data into different kinetics models. Table 2 showed data of different kinetic models with highest correlation coefficient value (R 2 = 0.9242) for Korsmeyer-Peppas followed by the Higuchi model (R 2 = 0.8572), First-

Evaluation of APL-Loaded NEs Chitosan Based Gel
The pH measurement govern skin compatibility with developed gel, suitable for topical application and free from allergic reaction and skin irritation. The pH values of ANE4-loaded gel was noted to be 6.8 ± 1.2, which is optimum for dermal application. The success of topical gel depends on the spreadability, it's an important parameter on which therapeutic efficacy relies. A small shear is required for uniform spreadability of gels to the skin [28]. Spreadability of the developed NE4-loaded gels exhibited the optimal spreadability over the plexy glass plates the area of formulation spread was observed as be 8.6 cm, suggesting practically excellent spreadability.

Diffusion Study and Release Kinetics Models
Comparative in-vitro diffusion study was performed for pure drug (APL), optimized NEs (NE4) and ANE4-loased gel ( Figure 6). Cumulative percentage drug release was found 18.79% from pure APL suspension, 54.88% and 84.17% from optimized NEs (ANE4) and ANE4-loaded gel after 24 h, respectively. An improved drug release was observed from developed topical gel due to its nanosize carrier, which could facilitate drug penetration across the dermal layer and improve therapeutic concentration at the local site [29]. The release kinetics was also determined by fitting the data into different kinetics models. Table 2 showed data of different kinetic models with highest correlation coefficient value (R 2 = 0.9242) for Korsmeyer-Peppas followed by the Higuchi model (R 2 = 0.8572), First-order (R 2 = 0.8265), and Zero order (R 2 = 0.6162). Among all fitted models, APNE4loaded chitosan gels release pattern was best fitted to Korsmeyers-Peppas model with release exponent (n) ≤ 0.45 indicating Fickian diffusion (case I diffusional) release mechanism. Drug release improved as NEs containing oil, surfactant and co-surfactant was incorporated into chitosan gel. Drug was released from NE4-loade chitosan gel by erosion mechanism [30,31]. Free drug-loaded NEs droplets could be easily permeated at a higher rate into the skin layers. order (R 2 = 0.8265), and Zero order (R 2 = 0.6162). Among all fitted models, APNE4-loaded chitosan gels release pattern was best fitted to Korsmeyers-Peppas model with release exponent (n) ≤ 0.45 indicating Fickian diffusion (case I diffusional) release mechanism. Drug release improved as NEs containing oil, surfactant and co-surfactant was incorporated into chitosan gel. Drug was released from NE4-loade chitosan gel by erosion mechanism [30,31]. Free drug-loaded NEs droplets could be easily permeated at a higher rate into the skin layers.

Stability Study
Cumulative % drug release at the 24th h was found to be 82.24% from ANE4-loaded gel, and after the shelf-life study period, it was found to be 81.41 ± 2.38%. The similarity factor was found to be 51.42, and drug content 97.16 ± 2.65% in the ANE4-loaded gel formulation. Insignificant variation in these parameters indicates developed gel was stable.

Anti-Inflammatory Studies
The anti-inflammatory efficacy of ANE4-loaded chitosan gel was compared with Diclofenac sodium gel (1%) using rat paw edema method (Figure 7). The developed ANE4loaded chitosan gel caused 40

Stability Study
Cumulative % drug release at the 24th h was found to be 82.24% from ANE4-loaded gel, and after the shelf-life study period, it was found to be 81.41 ± 2.38%. The similarity factor was found to be 51.42, and drug content 97.16 ± 2.65% in the ANE4-loaded gel formulation. Insignificant variation in these parameters indicates developed gel was stable.
of treatment. The differences in the % inhibition by ANE4-loaded chitosan gel were statistically highly significant (* p < 0.01) at 1, 3 and 4 h and statistically significant (* p < 0.05) at 2 h. Based on data, it can be observed that the ANE4-loaded chitosan gel formulation exhibited higher anti-inflammatory activity in comparison to standard Diclofenac sodium gel, probably due better efficacy.

Histopathology Study
Negative control skin biopsy after H&E staining as well as Masson's trichrome staining showed less volume of fibroblast and blood vessels compared to the standard control. However, the wound treated with the ANE4-loaded chitosan gel showed increased volume of fibroblasts and more regeneration of new blood vessels linked together with microvascular network. Inflammatory infiltration was more in the test, moderate in standard control-treated and lowered in the negative control, Figure 11. Masson's trichrome negative control slides showed mild, thin (standard control) and thick collagen deposition at the wound center, Figure 11, respectively. In the test group skin tissue represents thicker collagen, well vascularization well stratified epidermis and hair follicles formation compared to the negative and standard controls. The inflammatory response was observed in negative control but relatively less in standard control and reduced in test specimen. Verhoeff special staining slides of Negative control (untreated) wound tissue showed in Figure 11, poor wound healing rate as indicated with the persistent inflammation marked by necrotic tissue and little proliferation at edges of a wound. Standard control (Silver sulphadiazine 1% cream) showed in Figure 11, marked re-epithelialization at wound edges indicates; granulation tissue formation evidenced by fibroblast proliferation and collagen synthesis. Figure 11, represents the wound healing activity, the center of the wound not fully healed. Test (ANE4-loaded chitosangels) treated wound tissue exhibits increased collagenation and re-epithelialization, indicative of accelerated wound healing rate ( Figure 11). Histopathological study of exercised wound tissue showed improved wound healing rate indicated fibroblasts proliferation, neovascularization, epithelial regeneration, and collagen deposition activity in the test as compared to the negative (untreated) and standard control (Silver sulphadiazine 1% cream).

Histopathology Study
Negative control skin biopsy after H&E staining as well as Masson's trichrome staining showed less volume of fibroblast and blood vessels compared to the standard control. However, the wound treated with the ANE4-loaded chitosan gel showed increased volume of fibroblasts and more regeneration of new blood vessels linked together with microvascular network. Inflammatory infiltration was more in the test, moderate in standard controltreated and lowered in the negative control, Figure 11. Masson's trichrome negative control slides showed mild, thin (standard control) and thick collagen deposition at the wound center, Figure 11, respectively. In the test group skin tissue represents thicker collagen, well vascularization well stratified epidermis and hair follicles formation compared to the negative and standard controls. The inflammatory response was observed in negative control but relatively less in standard control and reduced in test specimen. Verhoeff special staining slides of Negative control (untreated) wound tissue showed in Figure 11, poor wound healing rate as indicated with the persistent inflammation marked by necrotic tissue and little proliferation at edges of a wound. Standard control (Silver sulphadiazine 1% cream) showed in Figure 11, marked re-epithelialization at wound edges indicates; granulation tissue formation evidenced by fibroblast proliferation and collagen synthesis. Figure 11, represents the wound healing activity, the center of the wound not fully healed. Test (ANE4-loaded chitosangels) treated wound tissue exhibits increased collagenation and re-epithelialization, indicative of accelerated wound healing rate ( Figure 11). Histopathological study of exercised wound tissue showed improved wound healing rate indicated fibroblasts proliferation, neovascularization, epithelial regeneration, and collagen deposition activity in the test as compared to the negative (untreated) and standard control (Silver sulphadiazine 1% cream).

Conclusions
In this study, eucalyptus oil based NEs was developed as a nanocarrier; optimum APL-loaded NEs (APNE4) was then impregnated into chitosan gels for enhanced antiinflammatory and wound healing activity. Optimized NEs (ANE4) containing EO (14%, w/w) as the oil phase and Tween-80 (20%, w/w), THP (40%, w/w) as surfactants and cosurfactants, respectively, were subjected to physicochemical characterization and pharmacological evaluations. In vitro release data showed 2.91 and 4.47 fold increase in APL release with sustained pattern from optimized NE (ANE4) and ANE4-loaded chitosan gel in comparison to pure APL drug suspension. Superior anti-inflammatory activity and improved wound healing efficiency were exhibited by ANE4-loaded chitosan gel in comparison with the negative and standard controls. This formulation is expected to improve therapeutic effectiveness and patient compliance with less systemic toxicity.

Materials
Apremilast (APL) was purchased from Beijing Mesochem Technology Co., Ltd., China. Kolliphore EL, Eucalyptus oil, Olive oil Tween 20 and Tween 80, Ethyl alcohol were procured from Sigma Aldrich, St. Louis, MO, USA. Lauroglycol 90, Capryol PGMC, and Transcutol HP were obtained from Gattefosse, Lyon, France. Ultrapure water collected from the Milli-Q Water purification system was used throughout the study. All the reagents and chemicals of analytical grades were used without any further purification and process.

Determination of APL Solubility in Oils, Surfactants, and Co-Surfactants
Solubility studies of APL were performed as described previously reported method [33] with some modification. Briefly, an excess amount of APL drug was added in 1.5 mL

Conclusions
In this study, eucalyptus oil based NEs was developed as a nanocarrier; optimum APL-loaded NEs (APNE4) was then impregnated into chitosan gels for enhanced antiinflammatory and wound healing activity. Optimized NEs (ANE4) containing EO (14%, w/w) as the oil phase and Tween-80 (20%, w/w), THP (40%, w/w) as surfactants and co-surfactants, respectively, were subjected to physicochemical characterization and pharmacological evaluations. In vitro release data showed 2.91 and 4.47 fold increase in APL release with sustained pattern from optimized NE (ANE4) and ANE4-loaded chitosan gel in comparison to pure APL drug suspension. Superior anti-inflammatory activity and improved wound healing efficiency were exhibited by ANE4-loaded chitosan gel in comparison with the negative and standard controls. This formulation is expected to improve therapeutic effectiveness and patient compliance with less systemic toxicity.

Materials
Apremilast (APL) was purchased from Beijing Mesochem Technology Co., Ltd., China. Kolliphore EL, Eucalyptus oil, Olive oil Tween 20 and Tween 80, Ethyl alcohol were procured from Sigma Aldrich, St. Louis, MO, USA. Lauroglycol 90, Capryol PGMC, and Transcutol HP were obtained from Gattefosse, Lyon, France. Ultrapure water collected from the Milli-Q Water purification system was used throughout the study. All the reagents and chemicals of analytical grades were used without any further purification and process.

Determination of APL Solubility in Oils, Surfactants, and Co-Surfactants
Solubility studies of APL were performed as described previously reported method [33] with some modification. Briefly, an excess amount of APL drug was added in 1.5 mL of oils (Kolliphore EL, Lauroglycol 90, Eucalyptus oil, Capryol PGMC and Olive oil), surfactant (Tween 20 and Tween 80), and co-surfactants (Ethyl alcohol and Transcutol HP) in a glass vial, and vials were tightly capped and kept on biological shaker "(LabTech, LBS-030S, Kyonggi, Korea)" for 72 h at 37 ± 2 • C. The samples were centrifuged at 6000 rpm "(Hermle-Labortechnik, Z216MK, Wehingen, Germany)" for 15 min. The supernatant was diluted with methanol appropriately and analyzed for drug content by UV/Vis spectrophotometer at wavelength 229 nm [27]. Based on the highest solubility, selected oil, surfactant, and co-surfactant were further used to construct the ternary phase diagram.

Pseudo-Ternary Phase Diagram
Based on solubility studies of APL in different components, distilled water (aqueous phase), Eucalyptus oil (oil), Tween-80 (surfactant), and THP (co-surfactant) were used for the construction of the ternary phase diagram. The surfactant and co-surfactant (Smix) at different weight ratios (1:0, 1:1, 1:2, and 2:1) were used to prepare the triple-phase diagram. Oil and Smix were thoroughly mixed at ratio 1:9 to 9:1 and titrated with distilled water till the formation of hazy dispersion [34,35].

Selection and Preparation of APL-Loaded NEs
Different formulations compositions were selected from the region of triple-phase diagrams to incorporate the APL drug (Table 3). Prepared nanoemulsion was enclosed in the screw vial and placed at room temperature to check the phase separation. Accurately weighed drug (1%, w/w) of the total weight of APL was dissolved in eucalyptus oil, vortexed (Vortex 3, IKA ® -Werke GmbH & Co., Staufen, Germany) for 5 min, followed by addition of Smix composed of surfactant (Tween-80):co-surfactant (Transcutol HP), and stirred on a magnetic stirrer with a magnetic bar at room temperature until the dissolution of the drug. Then weighed amount of water (milli-Q) was added dropwise with continuous vortexing until isotropic bluish transparent systems were obtained, called NEs.

Physicochemical Properties of Developed APL-Loaded NEs
The developed NEs (ANE1-ANE5) were evaluated for mean droplet size, polydispersity index (PDI) and zeta potential (ZP) using "Malvern zetasizer (ZEN-3600, Malvern Instruments Ltd. Worcestershire, UK )". The NEs sample was diluted with deionized water (1:200) and transferred into plastic cuvette in order to acquire dispersion, sonicated for 10 min to break the coalescence, then mean droplet size, PDI and ZP were measured by passing the laser at an angle of 90 • at 25 • C operated by photon correlation spectroscopy [36,37]. The refractive index (RI) of APL-loaded NEs were determined using Abbe's Refractometer "(Precision Testing Instruments Laboratory, Germany)" [38,39]. Percentage of transparency of the NEs was measured by homogenizing the sample with methanol, transmittance (%) was measured by UV-spectrophotometer at λmax 650 nm using methanol as blank [40].

Thermodynamic Stability Study
Developed APL-loaded NEs (ANE1-ANE5) were exposed to six cooling/heating cycles between 4 • C and 45 • C for 48 h. The NEs that did not show any phase separation, creaming, or cracking, were further tested for accelerated stability by keeping the sample to freeze-thaw cycles for 48 h at each temperature −21 and +25 • C [41]. NE was further subjected for centrifugation test by giving rotation at 3000 rpm for 20 min.

Morphology
The optimized NEs (ANE4) was diluted with milli-Q water (1:2 v/v), and coated on the carbon grid. The paper absorbed excess fluid, moisture also removed by placing it in the desiccator for about two hours before the analysis. Droplet morphologic characteristics were captured using JEM-1010 transmission electron microscope "(JEOL, Tokyo, Japan)" operated at 200 kV.

Drug Excipient Interaction FTIR Study
The FTIR spectrums of pure APL and optimized NES (APNE4) were taken by using FT-IR Spectrometer (Thermo Science iD5 ATR diamond Nicolet iS 5 FT-IR Spectrometer, Waltham, MA, USA). The samples were examined at the wavenumber range of 4000-400 cm −1 [42].

Formulation of NE-Loaded Gel
Chitosan gel (2%, w/v) was prepared by dispersing chitosan in distilled water. The polymeric dispersion was then kept in the refrigerator overnight after adding 2-3 drops of triethanolamine for soaking and swelling of the polymer. The optimized NE4 was incorporated into chitosan gel under magnetic stirring at a speed of 300 rpm for a 24 h at room temperature [43].

Spreadability
The spreadability and rheological flow of gel is an essential parameter for patienttherapeutic compliance. The APNE2-loaded gel (0.5 gm) was placed between two plexus glass plates to measure spreadability. A circle (1 cm) marked on the external outer surface of plates, a weight of 500 gm was then placed on the top of the plate, and a sample was allowed to spread for 5 min. Increased in diameter due to the spreading was noted [44].

Diffusion Study and Release Kinetics Models
A diffusion study was carried out to plot the cumulative graphs and data utilized for release kinetics modeling. Franz diffusion cell of 10 mL capacity was used, filled with phosphate buffer (pH 7.4). The donor compartment was fixed with a dialysis membrane, molecular cut-off weight 14000 Da m (Sigma-Aldrich Co., St. Louis, MO, USA) and added with pure APL, optimized nanoemulsion (ANE4), and ANE4-loaded gel each containing 10 mg equivalent APL drug. The assembly was placed on a thermostatically controlled plate, acceptor compartment stirred by magnetic bead for 50 rpm maintained at 37 ± 0.5 • C [38]. At predetermined time intervals, samples were withdrawn, and the same volume was added with a fresh medium to replenish/sink condition. Samples were analyzed at 229 nm [27] using the UV-spectrophotometric method.
Release kinetics models; zero order, first order, Higuchi and Korsmeyer-Peppas model. Based on the regression analysis, the best model is proposed [35].
Zero-order Equation (1). Q t = amount of drug released at time t, Q 0 = initial drug amount in solution, and K 0 = Zero Order kinetic constant. First-order Equation (2). Q t = amount of drug released at time t, Q 0 = initial drug amount in solution, and K t = First Order release constant. Higuchi Equation (3) and Korsmeyer Peppas model Equation (4). Where K is a kinetic constant and n is the drug release exponent. If n ≤ 0.45 then the drug release follows the Fickian diffusion (case I diffusional), 0.45 < n < 0.89 referred to anomalous (non-Fickian) diffusion.

Stability Study
APNE4 loaded chitosan gel was assessed for a stability of gel. Briefly, developed gel was kept at 25 ± 2 • C/75 ± 5% RH for six months, particle size and drug content were measured in order to check any significant change. Diffusion study was performed as per the above-mentioned procedure, release data then fitted to the similarity factor equation-5 and f2 value was noted. As per the SUPAC, if the f2 value ranges >50 [45], the release pattern is considered identical, and the product reflects stability.
where: f 2 is a similarity factor; n, w t , R t , and T t are number of observations, optional weight, reference-APNE2 gel release (%), test-APNE2 gel-releases (%) after storage, respectively.

Animals
Healthy male Wistar rats (200-220 g) were received from the animal care unit of College of Pharmacy, Prince Sattam Bin Abdulaziz University, Alkharj, Saudi Arabia, and used to assess the Anti-inflammatory study and wound healing activity of gels. The rats were housed under standard laboratory of light and dark cycles, at 22 ± 2 • C and provided with standard diet. The Bioethical Research Committee approved the animal studies" (BERC-008-04-21)" Prince Sattam bin Abdulaziz University, Al-Kharj, Saudi Arabia.

Anti-Inflammatory Study
Anti-inflammatory studies of prepared gel formulation was compared to fusidic acid gels using rat paw edema method [46]. Single dose parallel design was used for the antiinflammatory studies. Twelve rats were randomly selected and divided into three group containing four animals (n = 4) each. Inflammation in the left hind paw of each rats were induced by injecting 0.1 mL (1%, w/v) carrageenan in normal saline. Animals in Group-I rats 1% CMC (NC) on pantar surface of left hind paw, rats of group II and group III received ANE4-loaded chitosan gel and Diclofenac sodium gel (1%), respectively. The rat paw volume of each rat was measured at 0, 1, 2, 3 and 4 h post injection of carrageenan using plethysmometer. The percent edema inhibition was calculated using following equation: % Paw Edema Inhibition = (edema control rats-edema in treated rats)/(edema in control group) × 100

Wound Healing-Excision Model
Developed ANE4-loaded gel was investigated for wound healing activity by excision models. All the rats grouped in three categories were anesthetized using ketamine (25 mg/kg) and xylazine (10 mg/kg), intraperitoneal injection. Hair clipper was used to shave the dorsal thoracic side, the cutaneous approximately circular wound of 230 mm width with 2 mm depth was inflicted using a surgical scissor. Rats were fed with the standard pellets and water ad libitum; they kept undressed to the open aseptic condition. The wound was then cleaned with normal saline (0.9% w/v, NaCl solution); after complete hemostasis, treatment was started; negative control (non-therapeutic), standard control (1% w/w, silver sulfadiazine cream) [47], test (ANE4-loaded chitosan gel) were applied to cover the wound every day until complete healing. Wound diameter was measured by using the OHP sheet at 1, 7, 14 and 21 days until complete epithelialization took place and with resulted in wound closure [48,49]. The data from all the groups were assessed to take the mean and Standard deviations. Results were used to calculate the area of the wound, wound healing, and percent decrease in the wound size.
Wound contraction (%) was calculated using the following equation.

Histopathology Study
Skin tissue section of (3-5 cm) from three animal groups (negative control, standard control, and test) were collected into a suitable plastic closed container filled with 50 mL of 10% formalin (Sigma-Aldrich, MICH, USA) solution and processed in automated tissue processing machine (Leica ASP300S Fully Enclosed Tissue Processor, IL, USA). The processed tissue sample was embedded in paraffin, 5 µm thick section was prepared using a rotary microtome (SHUR/Cut 4500, TBS, NC, USA). Three sections from each group were stained; first section was stained with hematoxylin-eosin (H&E). The second and third sections were stained with masson trichrome (MT) and Verhoeff's Hematoxylin (VH), respectively. Wound healing was observed under the microscope by expert histopathologists.

Statistical Analysis
"One-way ANOVA using Dunnett's test. However, an unpaired t-test was used for the statistical evaluation. The GraphPad InStat software (GraphPad Software, San Diego, CA, USA) was used for statistical analysis, and p < 0.05 and p < 0.01 was considered significant and highly significant respectively".