Box–Behnken Design: Optimization of Proanthocyanidin-Loaded Transferosomes as an Effective Therapeutic Approach for Osteoarthritis

Transferosomes are one of the vesicular carriers that have received extensive research and attention recently because of their capacity to get beyond the barriers posed by the stratum corneum to penetration. The intent of the current study is to optimize and evaluate proanthocyanidin (PAC) containing transferosomal transdermal gels. PAC-containing transferosomes were prepared using the film hydration method and then loaded into a 4% methylcellulose gel. A 23 Box–Behnken design was used to optimize the PAC-loaded transferosomal gel, where the effects of phospholipid 90 G (X1), Tween 80 (X2), and sonication time (X3) were evaluated. The formulation factors, such as the drug entrapment efficiency percentage (PEE) and in vitro drug release, were characterized. A PEE of 78.29 ± 1.43% and a drug release in vitro at 6 h of 24.2 ± 1.25% were obtained. The optimized transferosomal-loaded proanthocyanidin (OTP) formulation penetrated the porcine skin at an excellent rate (0.123 ± 0.0067 mg/cm2/h). Stability tests were conducted for OTP to predict the effects of various temperature conditions on the physical appearance, drug content, and PEE for periods of 15, 30, and 45 days. Finally, this transferosomal system for transdermal PAC delivery may be a suitable alternative to the conventional treatment for osteoarthritis.


Introduction
The German company IDEA AG has registered the word "transferosome" as a trademark, which is derived from the Greek word "soma", meaning "body", and the Latin word "transferre", meaning "to transport across" [1]. Lipid-based transferosomes are stiff lipid bilayers (liposomes) or non-ionic surfactant monolayer vesicles (niosomes) that are flexible, ultradeformable, and stress-responsive [2]. While the drug delivery systems such as nanoparticles, liposomes, and niosomes are applied to the skin, they typically can only penetrate the outermost layers of the stratum corneum (SC), accumulating in the epidermal layer, but are not able to penetrate deeper layers of the skin, such as the dermis, or reach systemic levels that are effective. Liposomes are commonly preferred drug delivery methods for cutaneous applications. According to published papers, small unilamellar liposomes have a higher skin penetration capability than bigger ones [3].
Despite being more durable and impervious to modifications in osmolarity than regular liposomes, niosomes' piercing capacity has been linked to lower fluxes through the SC [4]. When likened to liposomes and niosomes in liquid media, transferosomes have asserted the greatest colloidal stability in the form of zeta potential [5]. Transferosomes have The proanthocyanidin (PAC) was a gifted sample from Verax Life Science, Himachal Pradesh, India. Phospholipid 90 G (PC) was purchased from Lipoids Switzerland. Tween 80, sodium hydroxide pellets, and potassium dihydrogen orthophosphate were purchased from Hi-media Pvt Ltd., Mumbai, India. Ethanol and chloroform were procured from Changshu Hongshu Great Chemical Co Ltd., Mumbai, India. All the chemical compounds were of analytic grade (AR).

Compatibility Evaluation Using Fourier Transform Infra-Red Spectroscopy (FTIR)
To provide the optimum interaction, the drug and excipient ratio in the formulation of transferosomes was studied at a 1:1 proportion. Additionally, the interaction between the drug and excipient was verified by FTIR (Shimadzu IR Affinity, Kyoto, Japan) in the scanning range of 4000-400 cm −1 using the KBr technique [25].

Preparation and Optimization of PAC-Loaded Transferosomes
PAC-loaded transferosomes, adopting the thin film hydration approach, were formulated. In brief, PACs, lipids (phospholipid 90 G), and Tween 80 were dissolved in 40 mL of chloroform; when the chloroform evaporated at 120 rpm and 40 • C, a thin film was created in the rotary evaporator. To remove the extra solvent, a thin layer of lipids was dried for 10 min at 50 • C and under vacuum overnight. The dried thin film was then hydrated with phosphate-buffered saline (PBS) pH 7.4 for 30 min while being shaken at a temperature that was 10 • C above the amphiphiles phase transition temperature (T c ). The dispersion was allowed to fully hydrate for 4 h at room temperature, after which it was subjected to sonication for the predetermined number of minutes, as indicated in the design, in order to break the multilamellar liposomes [25] and transfer them to a 4% w/v methylcellulose gel.

Experimental Design
Phospholipid concentration (mg) (X1), Tween 80 (mL) (X2), and sonication duration (min) (X3) were the three main independent variables that were adjusted at two levels, a low level (−1) and a high level (+1), in a three-factor, two-level factorial design (2 3 ) for the optimization approach. Following preliminary tests, the values of two coded levels of three components were presumptive; they are displayed in Table 1. The dependent variables were the percentage entrapment efficiency (PEE) (Y1) and in vitro diffusion at 6 h (Y2) of prepared transferosomal gels containing PAC through skin. Design Expert 11 software was employed for the creation and assessment of the statistical experimental design. Table 2 displays the design's response matrix, which includes the permeation flux as a response.

Determination of Percentage Entrapment Efficiency (PEE)
Transferosomal suspensions were ultracentrifuged for 30 min at 20,000 rpm and 10 • C. A UV-visible spectrophotometer (UV 1800 Shimadzu, Kyoto, Japan) was used to detect absorbance at 278 nm after centrifugation, and 1 mL of the supernatant was diluted by the addition of 9 mL of phosphate saline buffer (pH 7.4) [26]. The efficiency of drug entrapment was determined [27] as follows: where PEE is the drug entrapment efficiency, W T is the total quantity of drug in the transferosomal suspensions, and W F is the free drug in the supernatants.
2.6. Size, Polydispersity Index, and Zeta Potential Transferosomes were assessed for particle size, polydispersity index (PDI), and zeta potential using a dynamic light scattering device (Zetasizer Nano; Malvern Instruments Ltd., Malvern, UK). For estimation, 1 mL of the formulation was introduced to the analyzer in the see-through Malvern zeta potential cuvette [28].

Scanning Electron Microscopy (SEM)
SEM was employed for the visualization of the vesicle morphology. A glass slide was covered with one drop of transferosomal preparation, which was then spread out and left to dry. The item was coated with gold using a cool spit coater after it had dried and was then visualized with a scanning electron microscope at a voltage of 10 KV [29,30].

Transmission Electron Microscopy (TEM)
TEM analysis was used to examine the surface morphology of manufactured transferosomes (Jeol, JEM-1010, Tokyo, Japan). Before being mounted into the microscope, a silicon wafer was coated with gold and air-dried at room temperature with a diluted suspension of transferosomes (transferosomes:water, 1:5) in Milli-Q water. The image was taken at a 5 kV accelerating voltage [31].

Drug Content
The vesicles were lysed by sonication for 15 min with 10 mL of methanol and 1 g of the transferosome gel formulation. Later, a centrifuge tube containing this solution was filled, and it was spun at 2000 rpm for one hour. A liquid supernatant was discarded, and dilution was applied as necessary. A UV spectrophotometer (UV 1800 Shimadzu) set to 278 nm was used to measure absorbance [32]. % Drug Content = Amount of drug obtained after centrifugation Amount of drug taken × 100 (2)

In Vitro Drug Diffusion Studies
Franz diffusion cells with a diffusional area of 3.14 cm 2 were used to conduct in vitro drug diffusion tests at a time interval of 1, 2, 4, and 6 h for all the formulations. In between the donor and receptor compartments, an egg layer [33] was placed. The formulation was applied to egg film. The phosphate buffer pH 7.4 in the receptor compartment was continuously mixed by a remotely controlled Teflon-coated magnetic bead. To imitate physiological conditions, the temperature of the cell was maintained at 37 ± 1 • C. A UV spectrophotometric approach was used to measure the concentration of the PAC in aliquots (1 mL each) throughout the course of 6 h at regular intervals. Similarly, the OTP was estimated for in vitro diffusion studies for 24 h at periodic time intervals. The results were fitted to various kinetic studies [34]. The diffusion study was carried out in triplicate.

Method for Egg Membrane Preparation
Egg shells had their contents taken out before being immersed for 30 min in diluted hydrochloric acid. The egg membrane was carefully detached and thoroughly cleaned with distilled water [35,36].

Preparation of Skin for In Vitro Skin Permeation Study
Porcine ear skin was purchased from the slaughterhouse within an hour after the animal was killed, prepared in accordance with the literature, and used in all permeation studies [37]. After using an animal hair clipper to remove the hair from the upper half of the skin surface, the entire thickness of the skin was gathered. By using a surgical scalpel, the fatty layer that was adherent to the dermis side was removed. The skin was then wrapped with aluminum foil after being cleaned with deionized water. The skin samples were used within a week after being kept at 20 • C.

Ex Vivo Skin Permeation Study
Utilizing a Franz diffusion cell, ex vivo skin permeation tests of the improved formulation were performed. The cell has two compartments: the donor and the receptor, each with a 1.43 cm 2 diffusion area. The donor compartment was exposed to the atmosphere and had a top opening. The stratum corneum of the resected pig ear skin was positioned to be donor-compartment-facing and clamped between the compartments of the diffusion cell. The receptor chambers were filled with the receptor phase, and magnetic stirrer bars were added. As a receptor medium, PBS (pH 7.4) was utilized. To stop any microbial growth, a very small amount of sodium azide (0.0025% w/v) was applied [38]. A magnetic stirrer was used to keep the temperature of the entire setup at 37 ± 0.5 • C. To help the skin samples hydrate, the skin sections were originally kept in the Franz cells for 2 h. Following this time frame, 5 mL of the OTP was applied to the skin's surface. Transferosomal gels were used in the examination of ex vivo skin permeability. Over the course of the investigation, 0.5 mL of medium was taken out of the receptor compartment at regular intervals and refilled with the same influx of buffer. Utilizing a UV-visible spectrophotometer, the amount of drug that had diffused was determined by scanning the absorbance at 278 nm.

Physical Stability of the Transferosomes
Stability tests were carried out for 45 days at various temperatures to ascertain the formulation transferosome vesicles' capacity to retain drugs under changes in temperature and relative humidity (RH). According to the recommendations of the International Conference on Harmonization (ICH), OTP was placed in a sealed vial and kept at 4 • C and room temperature for 15, 30, and 45 days. The physical appearance, drug content, and PEE were examined after samples were removed [39].

Results and Discussion
In the present investigation, to get around the primary problems with its oral distribution, PAC-loaded transferosomes were created, developed, and tested for their potential to be delivered transdermally. Specifically, non-toxic and biocompatible surfactant namely tween with phospholipid were used to study the transferosome gels.

Compatibility Evaluation Using Fourier Transform Infra-Red Spectroscopy (FTIR)
The FTIR spectra depict the combination of drug, tween 80, phospholipid, cholesterol, blank, and OTP. The characteristic peak in the FTIR spectra of pure PAC drug was found at 2978.09 cm −1 (dimer O-H), 1442.75 cm −1 (aromatic C-C ring), 1249 cm −1 (ester C-O stretch), 1357.89 cm −1 (alkanes C-H), and 972 cm −1 (=C-H out of plane). Tween 80 showed the characteristic peaks at 2862.36 cm −1 (alkanes CH stretch), 1735.77 cm −1 (C=O stretch), 1651.07 cm −1 (alkenes C=C stretch), 1458.18 cm −1 (alkanes CH 2 and CH 3 ). When the physical mixture of drug, Tween 80, and phospholipid 90 G was analyzed, the characteristic peaks of drugs were present in the physical mixture similar to that of an individual drug spectrum, and there were no discernable changes in FTIR spectra, which confirmed the absence of any chemical interactions between them [40,41], as shown in Figure 1. and room temperature for 15, 30, and 45 days. The physical appearance, drug content, and PEE were examined after samples were removed [39].

Results and Discussion
In the present investigation, to get around the primary problems with its oral distribution, PAC-loaded transferosomes were created, developed, and tested for their potential to be delivered transdermally. Specifically, non-toxic and biocompatible surfactant namely tween with phospholipid were used to study the transferosome gels.

Compatibility Evaluation Using Fourier Transform Infra-Red Spectroscopy (FTIR)
The FTIR spectra depict the combination of drug, tween 80, phospholipid, cholesterol, blank, and OTP. The characteristic peak in the FTIR spectra of pure PAC drug was found at 2978.09 cm −1 (dimer O-H), 1442.75 cm −1 (aromatic C-C ring), 1249 cm −1 (ester C-O stretch), 1357.89 cm −1 (alkanes C-H), and 972 cm −1 (=C-H out of plane). Tween 80 showed the characteristic peaks at 2862.36 cm −1 (alkanes CH stretch), 1735.77 cm −1 (C=O stretch), 1651.07 cm −1 (alkenes C=C stretch), 1458.18 cm −1 (alkanes CH2 and CH3). When the physical mixture of drug, Tween 80, and phospholipid 90 G was analyzed, the characteristic peaks of drugs were present in the physical mixture similar to that of an individual drug spectrum, and there were no discernable changes in FTIR spectra, which confirmed the absence of any chemical interactions between them [40,41], as shown in Figure  1.

Preparation and Optimization of PAC-Loaded Transferosomes
Using a Box-Behnken design, a three-factor, two-level, appropriate statistical tool (Design-Expert DX 11 software), and one-way ANOVA at 0.05 levels, a thorough investigation of the effects of process parameters such as phospholipid concentration (mg) (X1), Tween 80 (mL) (X2), and sonication time (min) (X3) and their interactions was the goal of using the experimental design. Since the Box-Behnken design demands minimum runs compared to a central composite design, it was deliberately chosen [42]. A design matrix was produced by the Design Expert software using data from 15 experimental runs. Table 2 displays the outcomes of the 15 formulations that were formulated. The three-dimensional plots for each of the three responses of Y1 and Y2 are displayed in Figure 2. These diagrams are well known for analyzing the interactions between variables and responses as well as for researching the combined effects of two factors on a response. Figure 3 shows a quantitative comparison between the experimental response values that were obtained and those that were anticipated. Table 3 displays the findings of the

Preparation and Optimization of PAC-Loaded Transferosomes
Using a Box-Behnken design, a three-factor, two-level, appropriate statistical tool (Design-Expert DX 11 software), and one-way ANOVA at 0.05 levels, a thorough investigation of the effects of process parameters such as phospholipid concentration (mg) (X1), Tween 80 (mL) (X2), and sonication time (min) (X3) and their interactions was the goal of using the experimental design. Since the Box-Behnken design demands minimum runs compared to a central composite design, it was deliberately chosen [42]. A design matrix was produced by the Design Expert software using data from 15 experimental runs. Table 2 displays the outcomes of the 15 formulations that were formulated. The three-dimensional plots for each of the three responses of Y1 and Y2 are displayed in Figure 2. These diagrams are well known for analyzing the interactions between variables and responses as well as for researching the combined effects of two factors on a response. Figure 3 shows a quantitative comparison between the experimental response values that were obtained and those that were anticipated. Table 3 displays the findings of the regression analysis for the

Entrapment Efficiency
Color points by value of

Entrapment Efficiency
Color points by value of   PEE is the portion of the whole drug (PAC) that is successfully contained within the transferosomes. Phospholipid 90 G has been successfully employed to create vesicular structures resembling liposomes since it is soluble in lipidic or aromatic hydrocarbon carriers but insoluble in polar solvents. Figure 2 displays three-dimensional response surface graphs related to PEE. Figure 3 displays the two-dimensional contour graphs relating to PEE. The three-dimensional response surface plot and the accompanying twodimensional contour plots show an increase in PEE, with increased Tween 80 up to a certain point before it was discovered to be reduced [43]; that increasing sonication time also increases PEE. Additionally, it was shown that the amount of PEE in transferosomes increased when phospholipid 90 G (X1) levels increased [44].
The model is suggested to be significant by the model F-value of 31.59. The likelihood of noise producing an F-value this large is only 0.01%. The predicted R 2 of 0.7613 and the adjusted R 2 of 0.9516 are reasonably in agreement; therefore, the difference is less than 0.2. The ratio of signal-to-noise is measured with adequate precision. Entrapment efficiency for PAC ranged from 77.29% to 85.81%, with an average of 80.52%. Figure 3 displays the linear correlation plots (C) between the actual and predicted values as well as the associated contour plots (D) for the responses.

Response 2 (Y2): Effect of Independent Variables on In Vitro Diffusion at 6 h
The in vitro drug diffusion profile at the time period of 6 h for the formulations is shown in Table 2. Due to the lipid's limited ability to hold substantial amounts of the drug, the free drug may have been disposed of near the surface, which might explain the existence of both free and entrapped drugs. This led to a quick initial release (caused by the presence of the free drug and the drug adsorbed on the surface), followed by a slower sustained release phase (caused by the diffusion of the entrapped drug through the lipid bilayers of the vesicles), which is very effective at sustaining and controlling the release of PAC. These results were in line with those of El Zaafarany et al. [45]. The release rate is significantly impacted by the sonication time.
As the drug flows through the bilayers of the vesicular structure and results in a decrease in release rate, it is well known that any factor that promotes the production of transferosomes or raises their PEE will impact the release rate [46]. When Tween 80 was used, the diffusion rate was accelerated as the solvent volumetric ratio increased. Additionally, when Tween 80 was used, the release rate was found to be slowed down by increasing the amount of medication administered, which may be related to an increase in PEE [47].

Numerical Point Prediction Method
By using the numerical point prediction optimization method of the software Design Expert 11, the ideal transferosomal formulation was chosen based on the criteria of achieving the acceptable value of the entrapment efficiency of PAC and in vitro diffusion at 6 h. The formulation, with phospholipid 90G (175.97 mg), Tween 80 (43.59 mL), and sonication duration (25.79 min), was determined to satisfy the requirements of an OTP formulation with a desirability value of 0.966. Scanning electron microscopy was used to examine the morphology of the optimized formulation. The image showed how the medication became tangled up in the lipid network. It displayed well-known round vesicles of a consistent size with a stable nano-vesicular shape. The entrapment efficiency of PAC was 78.29 ± 1.43%, while in vitro diffusion at 6 h for OTP formulation was 24.2 ± 1.25%. Investigative estimates of the PEE of PAC and in vitro diffusion at 6 h produced by the OTP were discovered to be in agreement with the predicted values of the PEE of PAC (79.03 ± 1.24%) and in vitro diffusion at 6 h (24.67 ± 1.65%) produced by the Design Expert software, indicating that the optimized formulation was plausible and reputable.

Size, Polydispersity Index, and Zeta Potential
The outcomes demonstrated that OTP dispersion significantly reduces particle size with a restricted size distribution. It was discovered that the OTP's average particle size was 65 ± 14.44 nm. Figure 4 shows the distribution of vesicle sizes for the optimized formulation. It was discovered that the improved formulation's zeta potential was −13.0 mv; prior studies had also revealed a value that was very comparable [48]. The value implies the high-level stability of the optimized formulation. Due to the use of Tween 80, these values indicate that the particles have a neutral charge (a non-ionic surfactant). Tween 80, a surface stearic stabilizer, is used to cover the surface of nanoparticles to stop them from aggregating. The formulation would be stable as a result. the OTP were discovered to be in agreement with the predicted values of the PEE of PAC (79.03 ± 1.24%) and in vitro diffusion at 6 h (24.67 ± 1.65%) produced by the Design Expert software, indicating that the optimized formulation was plausible and reputable.

Size, Polydispersity Index, and Zeta Potential
The outcomes demonstrated that OTP dispersion significantly reduces particle size with a restricted size distribution. It was discovered that the OTP's average particle size was 65 ± 14.44 nm. Figure 4 shows the distribution of vesicle sizes for the optimized formulation. It was discovered that the improved formulation's zeta potential was −13.0 mv; prior studies had also revealed a value that was very comparable [48]. The value implies the high-level stability of the optimized formulation. Due to the use of Tween 80, these values indicate that the particles have a neutral charge (a non-ionic surfactant). Tween 80, a surface stearic stabilizer, is used to cover the surface of nanoparticles to stop them from aggregating. The formulation would be stable as a result.

Scanning Electron Microscopy (SEM)
Optical microscopy was used to examine the surface morphology. SEM analysis revealed that the PAC transferosomes (OTP) were found to be merely in spherical forms. Optical microscopical and SEM images are shown in Figures 5 and 6, respectively.

Scanning Electron Microscopy (SEM)
Optical microscopy was used to examine the surface morphology. SEM analysis revealed that the PAC transferosomes (OTP) were found to be merely in spherical forms. Optical microscopical and SEM images are shown in Figures 5 and 6, respectively. proanthocyanidin.

Scanning Electron Microscopy (SEM)
Optical microscopy was used to examine the surface morphology. SEM analysis revealed that the PAC transferosomes (OTP) were found to be merely in spherical forms. Optical microscopical and SEM images are shown in Figures 5 and 6, respectively.

Transmission Electron Microscopy (TEM)
The formulated OTP was envisioned by TEM; the image is presented in Figure 7. The TEM image witnesses the presence of a multilamellar vesicular structure. Nonetheless, these lamellar vesicles were discovered to be spaced to the core [49].

Drug Content
The results obtained showed 90.28 ± 1.23% drug content in OTP, which shows no degradation of the drug in the process [31].

Transmission Electron Microscopy (TEM)
The formulated OTP was envisioned by TEM; the image is presented in Figure 7. The TEM image witnesses the presence of a multilamellar vesicular structure. Nonetheless, these lamellar vesicles were discovered to be spaced to the core [49].

Transmission Electron Microscopy (TEM)
The formulated OTP was envisioned by TEM; the image is presented in Figure 7. The TEM image witnesses the presence of a multilamellar vesicular structure. Nonetheless, these lamellar vesicles were discovered to be spaced to the core [49].

Drug Content
The results obtained showed 90.28 ± 1.23% drug content in OTP, which shows no degradation of the drug in the process [31].

Drug Content
The results obtained showed 90.28 ± 1.23% drug content in OTP, which shows no degradation of the drug in the process [31].

In Vitro Drug Diffusion Studies
In vitro drug diffusion of OTP was performed and observed in a UV spectrophotometer at the 278 nm range. The samples were withdrawn at predetermined intervals to determine the percent drug permeation after 24 h. The results were fitted to kinetic studies, such as the order of drug diffusion and Higuchi's and Peppa's plots. The OTP shows an R 2 value of 0.9974 for the in vitro diffusion study, which clearly indicates that the formulation obeys a zero-order kinetic pattern. The R 2 and n-value of Higuchi's and Peppa's plots were found to be 0.9424 and 0.9316, respectively, which indicate the release mechanism followed was a diffusion-mediated non-Fickian type. The in vitro drug diffusion plots are shown in Figure 8.

Ex Vivo Skin Permeation Study
Franz diffusion cells were used to test the ex vivo skin permeation of the designed OTP, utilizing excised pig skin as the permeation membrane. Over the course of 24 h, it was discovered that these transferosomal gels' ex vivo skin penetration of OTP was sustained ( Figure 9). These transferosomal gels' Jss (mg/cm 2 /h) was calculated to be 0.123 ± 0.0067 mg/cm 2 /h. Therefore, the Jss of the transferosomal gel increased as the PEE in transferosomes containing PAC increased. This agreed with earlier studies that found that the transdermal penetration of medication when the deformable vesicles were applied topically was ten times higher than that of conventional vesicles [50]. Deformable vesicles containing sodium deoxycholate may have caused the change in water concentration. It has also been shown that the inclusion of surfactants in the phospholipid vesicles promotes the production of membrane ripples. These ripples also provide the intermembrane connection location for membrane fusion [37].

Ex Vivo Skin Permeation Study
Franz diffusion cells were used to test the ex vivo skin permeation of the designed OTP, utilizing excised pig skin as the permeation membrane. Over the course of 24 h, it was discovered that these transferosomal gels' ex vivo skin penetration of OTP was sustained ( Figure 9). These transferosomal gels' Jss (mg/cm 2 /h) was calculated to be 0.123 ± 0.0067 mg/cm 2 /h. Therefore, the Jss of the transferosomal gel increased as the PEE in transferosomes containing PAC increased. This agreed with earlier studies that found that the transdermal penetration of medication when the deformable vesicles were applied topically was ten times higher than that of conventional vesicles [50]. Deformable vesicles containing sodium deoxycholate may have caused the change in water concentration. It has also been shown that the inclusion of surfactants in the phospholipid vesicles promotes the production of membrane ripples. These ripples also provide the inter-membrane connection location for membrane fusion [37]. the transdermal penetration of medication when the deformable vesicles were applied topically was ten times higher than that of conventional vesicles [50]. Deformable vesicles containing sodium deoxycholate may have caused the change in water concentration. It has also been shown that the inclusion of surfactants in the phospholipid vesicles promotes the production of membrane ripples. These ripples also provide the intermembrane connection location for membrane fusion [37].

Physical Stability of the Transferosomes
The monitoring of the physical appearance, drug content, and PEE under various storage circumstances for a duration of 0, 15, 30, and 45 days was done to determine the stability of the OTP formulation. Throughout the 45 days, no significant changes were seen in any of the parameters when examined at 4 • C and at room temperature (Table 4).

Conclusions
The transferosomes were fabricated by loading PAC using the film hydration method; the PAC was incorporated into a 4% w/v methylcellulose gel. The effects of three independent factors such as phospholipid 90 G, Tween 80, and sonication time on PEE and in vitro drug release at 6 h were analyzed as responses in the process of identifying the optimized formulation (OTP) using 2 3 Box-Behnken design-based response surface methodology. A skin permeation flux of 0.123 ± 0.0067 mg/cm 2 /h was demonstrated in ex vivo PAC permeation from the OTP through pig skin, which represents a further improvement. Hence, this highly deformable vesicle-based transferosome (OTP) was found to be successive progress in the delivery of PAC through the transdermal route.