Tocopherol succinate‐loaded ethosomal gel synthesized by cold method technique: Deeper biophysical characterizations for translational application on human skin

Tocopherols are well‐known antioxidant and moisturizing agent. Tocopherol succinate (TS) are widely used in many skin products especially used in anti‐aging and skin whitening product formulation.


| INTRODUC TI ON
In recent years, drug delivery of therapeutic agents has been revolutionized by encapsulating them into nanosized vesicular carrier systems. 1,2These smart systems were reported very effective allowing to overcome limitations and problems associated with conventional drug delivery systems, 3,4 increasing attracting much attention among scientists worldwide. 5ansdermal route is widely used to deliver chemical drugs and natural compounds to treat skin conditions (e.g., skin diseases, premature aging), because of its numerous advantages over oral (per os) route including, avoidance of first pass effects, decreased fluctuation in drug plasma level, (local) targeted delivery leading to lower side effects and increase efficiency, increased patient compliance. 6,7ter soluble drug and molecule cannot cross the stratum corneum barrier as the skin is composed of bundles of keratin, crosslinked protein and covalently-bounded lipid molecules. 4,8Poor penetration is the main disadvantage of transdermal drug delivery due to presence of stratum corneum layer which act as skin barrier. 7,9,10[13] Also, it is worth mentioning that the use of chemicals and surfactants reduce the integrity of skin cells and disturb the original shape and elasticity of the skin. 14,15hosomes are concentric bi-layered, soft, and malleable vesicles mostly used for transdermal drug delivery system for both local and/or systemic effects. 16These lipid nanocarriers were first time developed by Touitou et al., 1997. 10,17 Ethosomes are mainly composed of ethanol in high concentration (20%-45%), phospholipids (0.5%-5%), and water. 3,6,14Ethanol first interacts with lipid molecule in the polar head group region, enhances the penetration of malleable ethosomal vesicles by decreasing the transition temperature of the skin lipid, and interacts with stratum corneum cells of dermal layer. 16Thereby, by reducing the density of the stratum corneum skin lipid, the layer fluidity of soft vesicles increases which results in enhanced delivery of the active moiety into the deep skin layers. 4Therefore, a synergistic mechanism was established between ethanol molecule, ethosomal vesicles, and skin lipid for enhanced transdermal drug delivery. 16,18Fusion of ethosomal vesicles with skin lipid causing the release of drug molecules into deeper skin layers by opening new pathways for drug penetration and permeation. 6,19Commercialization of ethosomal technology has been started in 2000 and is getting increasing interest including by a large number of pharmaceutical industries.Thereby, many cosmeceutical firms are involved especially in ethosomes-based product formulations because of their effective delivery of active agents 3,14,20 Indeed, ethosomal nanocarrier systems are preferably used over other conventional liposomes because they enhance the drug delivery and the permeation rate through deep skin layers. 6Moreover, ethosomes are considered convenient towards eco-friendly manufacturing, handling, storage, and transportation. 3,12,21,22copherols are commonly known as vitamin E and it is very important component of skin. 23They play crucial roles in the physiological functions of the skin and maintain its strength. 13,23,24Tocopherol act as pro-oxidant, free radical scavenger, stabilizing the biological membrane by protecting against polyunsaturated fatty acids (PUFA) peroxidation initiation. 3,14,24Besides, tocopherols act as moisturizing agents, preventing trans epidermal water loss (TEWL), and regulate keratin turnover. 25[27] The aim of the study is to develop TSEG with increased entrapment efficiency (EE), stability, skin penetration, and retention into skin layers for safe and efficient topical application studies have been carried first in vitro, ex vivo, and then in vivo.

| Reagents
Tocopherol succinate (TS), Propylene glycol (PG) and Carbopol-940 was purchased from Sigma Aldrich.Triethanolamine (TEA) was obtained from AppliChem.Soy phosphatidylcholine 90G from Lipoid, and ethanol of HPLC grade were purchased from Sigma-Aldrich (CHEMIE GmbH, Heidolph).Double distilled water (ddH 2 O) was used throughout the experiment study.All chemicals used for preparation of ethosomes were of analytical standard.by varying the quantities of ethanol (25%-40%) and propylene glycol (PG).0.25% TS (drug) was first dissolved into ethanol, and lipid soluble ingredients (phospholipids) were subsequently added under continuous stirring at room temperature (RT). 3,14PG (acting as a penetration enhancer) was then added at RT into the nonaqueous phase.Then, both the nonaqueous phase mixture and the aqueous phase, which was kept in a separate vessel, were preheated in a water bath at 30°C.The preheated aqueous phase was added dropwise (using a syringe) into the nonaqueous phase mixture, under continuous stirring at 750 rpm for 5 min.This mixture was cooled at RT and allowed to develop ethosomes vesicles.The size of ethosomal NPs (vesicles) could be reduced by stirring at 750 rpm for 30 min. 14,23The resulting mixture was sonicated for 30 min.Desired size of ethosomes vesicles can be developed by extrusion method technique.The prepared TSE NPs were stored in refrigerator for further analyses.The composition of TSE NPs (TSE vesicles suspension) is shown in Table 1.The EE was determined for each formulation (See Section 2.4).TS6 formulation (TS6F) displayed the best EE (99.89%) and was then selected for subsequent analyses.

| Determination of percent entrapment efficiency (% EE)
Simple indirect method was used to calculate the percentage (%) of drug entrapped into the developed ethosomal vesicles.One milliliter of ethosomal vesicles (suspension) was centrifuged at 12000 rpm for 30 min to get a clear supernatant solution; this process was repeated thrice.The collected supernatant solution was diluted with freshly prepared 0.2 M phosphate buffer solution (1x PBS, pH:6.8) and then analyzed by UV-Vis spectrophotometry at the wavelength of 285 nm. 14The %EE was measured from the calibration curve by using following formula:

| Particles size (PS), polydispersity index (PDI), and zeta potential (ZP)
Zeta sizer along with dynamic light scattering (DLS) method were used to determine the average size (PS), the bio-dispersion (PDI), and the stability of colloidal disperse TS6 particles (ZP), respectively.PDI-value range and uniformity.ZP is the charged equilibrium potential between the dispersed particles, and it is essential for the colloidal particle's stability of TSE vesicles.

| Transmission electron microscope (TEM) studies
TEM is a sub-microscopic imaging technique of high-resolution which allows to view the material at nano scale and can construct fine 3D images of cell structure.A drop of the diluted test formulation (TS6F) was placed on a copper grid coated with carbon.The test sample was kept on grid for 1 min to air dried it at RT. 2% w/w phosphotungstic acid stain was dropped on a carbon coated copper grid containing test sample.Extra stain was removed by tissue paper and analysis was performed by TEM at 60 KV.The test sample was examined at different TEM magnifications to evaluate the morphology and surface characteristics of dispersed vesicles.

| Thermogravimetric analysis (TGA)
Thermogravimetric analysis study was performed using DTA to determine the thermal stability of ethosomes.The change in mass as a function of temperature (20-400°C) was assessed.

| x-Ray diffraction analysis (XRD)
The crystalline properties of TS (pure drug), physical mixture, components of the formulation, and TS6F (optimized test formulation) were assessed by powder x-ray diffraction analysis (PXRD) using CuKa as a source.Results was analyzed from graphs constructed between counts/second and degree 2θ according to Bragg's law, (2dsineθ = nλ).

| Fourier-transform infrared (FTIR) spectroscopy
FTIR was used to get the spectra of TS (pure drug), PG, Ethanol, Phospholipid and TS6 (optimized test formulation).Each spectrum was obtained in the range of 400-4000 cm −1 and transmittance percentage was in the range of 85%-100%.Test samples were analyzed at mode 16 scans/sample by zinc selenium mode ATR (attenuated total reflectance).The peaks obtained from each spectrum were evaluated for determining the interactions between formulation components and the stability of TS6F.

| Preparation of ethosomal gel
Ethosomal gel was prepared by using 1% W/V carbopol 940 as gelling agent.One gram of carbopol-940 was soaked in small amount of double-distilled water (ddH 2 O) for overnight to make carbopol solution followed by addition of aqueous ethosomal suspension without TS control gel, namely CEG) or aqueous ethosomal suspension of TS drug (TS6F), making the final volume to 100 mL.The obtained mixture was stirred homogenously at 800 rpm by using overhead stirrer for 2 h until homogenous semi-solid consistency mass was obtained.0.9% w/v TEA solution was added dropwise and stirred continuously until homogenous gel with pH 6.8 was obtained.pH was adjusted to skin pH by using pH metry.Prepared gels (TSEG6 and CEG) were stored in refrigerator for further studies.

| In-vitro and ex-vivo evaluations of TSEG6
The developed ethosomal gels (TSEG6 and CEG) were evaluated in-vitro for potential organoleptic changes in terms of color, odor, appearance, feelings, pH, rheological behavior, and ex vivo for skin permeation.
For rheological assessments, viscometer was programmed at where, J = Steady state flux, ER = Enhancement ratio.

| In vivo study
A noninvasive, single blind and monocentric in-vivo study was con- (1)

| RE SULTS AND D ISCUSS I ON S
Tocopherols are lipophilic and act as naturally membrane-bound antioxidant.TS elicits moisturizing and antiaging properties. 3The major problem associated with TS is its sensitivity to light, oxidants, moisture and degradation by continuous UV exposure. 25,28,29TS can be added into skin lipid bilayer to protect the lipid peroxidation and act as barrier to prevent WL from skin layers.One of the previous study proved that TS has been used to prevent scars and heals lesions. 3,14TS has high molecular weight with highly lipophilic nature. 14,30For this reason, TS is formulated as nanocarrier system and added into semisolid system to develop TSEG formulations, proved promising for efficient and controlled drug delivery.Importantly, both hydrophilic and lipophilic, but also amphiphilic drugs, can be delivered by ethosomes into deep skin layers for local and systemic effects are achieved. 5,31rein, TSEG and CEG formulations were prepared successfully.Stable ethosomes were prepared by changing ethanol and phospholipid concentration.30% Ethanol and 0.5% phospholipid were optimal concentrations to develop vesicular membranes, enhance stability and EE of the TSEG.1% Carbopol-940 was used as jellifying agent.The concentration of ethanol used in classical ethosomes is 10%-50%. 32,33[36] Phospholipid act as penetration enhancer, it has effect on vesicles size but no effect on entrapment efficacy and used 0.5%-5% concentration. 3,13,14,37Propylene glycol (PG) is generally used in concentration 5%-20%. 32,38It acts as a membrane penetration enhancer and influences EE, vesicles size, and stability.Vesicles size decreases by increasing the PG concentration.The drawback of poor penetration, and instability issues during storage by niosomes have been overcome by the development of ethosomes. 5,39EG6 was selected as the optimized TSEG formulation based on its high EE (99.89%), and physicochemical stabilities.Ethosomes were prepared by the simple and easy-handling cold method. 3,14 this method, the aqueous phase was added dropwise carefully into the nonaqueous phase to produce ethosomes. 13,14Then, the size of developed ethosomal vesicles was further reduced by sonicating at RT, Nano sized vesicles are more stable and showed good release behavior from the developed formulation. 17,40,3TSEG6 was used for further in-vitro (e.g., physical characterizations.organoleptic changes), ex vivo (i.e., rat skin permeation) and in-vivo (i.e., patch test on humans) analyses.

| Entrapment efficiency (EE)
The percentage EE was more than 90% in all prepared ethosomal formulations.TSEG6 formulation showed the highest EE with 99.89 ± 1.32% whereas TSEG1 displayed the lowest EE with 93.88 ± 1.32% (Tables 1 and 2).The enhanced % EE is related to CH-stabilized bilayers and increased the thickness of vesicles bilayer, which prevent leakage of vesicular bilayer membrane. 41,42rthermore, increasing CH did not enhanced %EE due to competition between drug and cholesterol to stay within the ethosomes bilayer vesicles. 43

| Particle size (PS), PDI (polydispersity index), and Zeta Potential (ZP)
The size of the vesicular ethosomes were found by TEM in the acceptable range of 140.1-310 nm (Table 2).Also, they were uniform homogenized particle size distribution with reduced variation in PDI (0.114-0.250) (Table 2).Smaller sized ethosomes vesicles are produced as compared to niosomes vesicles.

| Transmission electron microscope (TEM)
TEM analysis technique widely used to direct measure the structure crystallography, particle size, size distribution, determine chemical composition, and morphology of dispersed vesicles. 5,14,17,44M confirms the morphology and surface characteristics of the developed ethosomes as spherical, smooth, round shape, and homogenous dispersed vesicles.Considering TSEG6 as optimized and selected formulation, the size of the ethosomal vesicles (or vesicular ethosomes) was about 250.1 nm (±1.53 nm; Figure 1).

| Thermogravimetric analysis (TGA)
TGA was performed to determine the thermal stability of TS (pure drug), PG, phospholipid, physical mixture (CEG, control formulation), and TSEG6 (test formulation) using a range of temperatures (0-35°C).Thermograms were constructed between temperature (°C) and % WL to evaluate the data (Figure 2).
The % WL occurred at reduced temperature in physical mixture due to the different material present.However, the ingredient melting point was different from each other.The decrease in melting peaks was produced in combined effect. 1 The formulation peak from the graph (Figure 2) showed the same range and it confirms the absence of any interaction among ingredients and ensure thermal stability of the developed formulation. 1,44

| Powdered x-ray diffraction (PXRD)
PXRD was assessed to evaluate the crystalline properties of TS pure drug, physical mixture CEG (control gel formulation), components/ingredients of the formulation, and TSEG6 (test gel formulation) (Figure 3).Graph was constructed between intensity (counts/ second) and degree 2θ according to Bragg's law, (2dsineθ = nλ). 45TS showed fused peaks representing amorphous nature and large peaks indicating crystalline nature.Crystalline nature of TS and physical mixture was observed distinctly through peaks. 1,45Whereas amorphous state of TS formulation indicated physical stability of TS in developed formulation. 45

| Fourier-transform infrared (FTIR) spectroscopy
FTIR spectra were obtained for TS drug, PG, ethanol, phospholipid and TSEG6 formulation (Figure 4).The FTIR peaks were analyzed to determined potential interactions between the formulation components/ingredients and TSEG6 formulation.
The peaks at 1366.67 cm −1 , 1377.14 cm −1 , 1368.12 cm −1 are at- TSEG6 was white in color while CEG was transparent in color and odorless when prepared freshly with optimal viscosity.Also, there were no temperature-dependent changes (p > 0.05) overtime in color, odor, smoothness, and pH.Although a slightly change in pH and flow properties of gels occur at higher temperature (40°C vs. 40°C ± 75% RH), the change was insignificant. 3,14Although pH values were slightly (p > 0.05) decreasing overtime when both gels were kept at one of the settled temperature, the change was insignificant. 3,14,48eological behavior of both fresh gels shows insignificant shear thinning effects at RT and follow non-Newtonian flow behavior (Figure 5).40°C and 40°C ± 75%.Decrease in pH values was not time-dependent (p > 0.05), but was temperature-dependent (p < 0.05).Table 3.
pH values for test and control formulation kept at 8°C, 25°C, 40°C, and 40°C ± 75% RH and Table 4. Physical stability analysis of TSEG6 (F) and CEG (C) at 8°C, 25°C, 40°C and 40°C ± 75%RH kept for the study duration of 3 months.
Skin permeation analysis conducted for topical preparations provides various important information.Wistar rat's abdominal skin was used to evaluate the permeation capacity of TSEG6 (test formulation) and CEG (control formulation) at pH 6.8 and 37°C 13 (Figure 6).From the kinetic modeling data, it can be noted that TS drug release from TSGE6 followed Fick's law of diffusion and exhibit a significative 46.7% increased permeation rate as compared to CEG permeated 22.2%. 14,18,24Furthermore, TSEG6 followed Krosmeyer-Peppas model and its regression coefficient value is R 2 = 0.9989 (Table 4).
Overall, due to good adhesive and hydrophilic property of carbopol 940, good physical stability characteristics, as well as effective increased permeation, TSEG6 can be effectively used for topical delivery. 1,13,24The statistical analysis depicted the less similarity and high difference in permeation rates as TSEG6 showed enhanced permeation as compared to CEG, significant results was obtained (p < 0.05; Table 5).

| In vivo study
The

F I G U R E 6
Comparison of percentage permeation of TSEG6 and CEG.
skin erythema, melanin level, (ii) moisture content, (iii) sebum level, and (iv) skin elasticity change was measured by mexameter (i), corneometer (ii), sebumeter (iii), and elastomer instrument (iv), respectively.In vivo study was performed by using noninvasive advanced devices equipped with sensitive probe.Skin sebum content, melanin, erythema, moisture content and elasticity level were evaluated with highly sensitive instrument with noninvasive probe.Human volunteers were selected after approval from Pharmacy Human ethics committee (PHEC), Ref.No.110-2020-/PHEC.

| Patch test
Patch test is very important for dermal drug delivery system and cosmetic products application.This test is noninvasive and used to analyze the skin hypersensitivity reaction caused by any ingredient of formulation. 14In the present study, both TSEG6 and CEG were applied on the right (circled as 'R') and left (circled as 'L') forearms, respectively.About 1 gm of TSEG6 and CEG were applied on selected area of 4 × 5 cm on each encircled forearm for respective formulation.
Patches were covered with surgical dressing in order to avoid the loss of applied formulation.Patch test was performed for 48 h.After not finding any hypersensitivity reactions, the in-vivo study was continued for 12 weeks.All volunteers were informed to apply formulation on their clean and washed face at night.They were also informed to come at prescheduled time for measurements and analyses for 12 weeks.They were also informed not to apply any other formulations and cosmetic product during this study period.Patch test was assessed by monitoring the skin melanin and erythema level change after application of both developed formulations, but also the moisture content, the sebum level, and the skin elasticity performance.

| Skin erythema and melanin
A mexameter equipped with highly sensitive noninvasive probe and multiple adopters, was used to measure the skin melanin index and erythema level just by placing the probe on the skin carefully (Figures 7 and 8).Skin erythema and melanin levels decreased (averaged values ± STD for each time point) continuously from Day 1 to 90th day of the study period after application of TSEG6, whereas they decreased in an irregular manner by application of CEG.All volunteers' obtained results were measured as mean % changes during the whole study period.Paired sample t-test applied between data obtained from TSEG6 and CEG formulations clearly displayed significant results (p < 0.01).This is due to the fact that tocopherol and its derivatives act as potent antioxidant agents and enable the reduction of melanin production. 49A study has proved that tocopherols act on the skin as antioxidant by modulating enzyme activity like tyrosinase, making them useful as whitening agents in cosmetics. 49,50Melanin is a skin pigment that gives color to skin.High concentration and accumulation of melanin causes pigmentation of skin due to UV exposure. 51High activity of tyrosinase enzyme causes the hyperpigmentation of skin. 52avonoids, phenolic content and antioxidant agent reduced the tyrosinase enzyme activity and decrease the melanin level in skin. 51,52Another study has reported that melanogenesis can be prevented by reducing the reactive oxygen species (ROS) level 53 in melanin-producing cells by use of antioxidants and tocopherols act as a good antioxidant agent.and its derivatives are used widely as skin moisturizing agent and in many cosmetic products. 13They act by preventing WL from dermal and epidermal skin layers. 56,57Tocopherols helps to maintain skin water content balance.In this formulation, PG and lipid were used that also produced skin occlusive effects and prevent WL from skin layers. 3,13,56

.8.4 | Skin elasticity
Elasticity occurs when skin elastic fibers gain original position and form after removal of applied stress.Skin collagen fiber and elastic fibers are responsible for skin elasticity. 3,58The aging process causes skin rigidness and reduction of skin elasticity, especially when the skin is exposed to continuous UV exposure (by sunlight) and/or contaminated with chemical substances. 59Tocopherols act as good moisturizing agents, antioxidants and help to maintain the health of skin cells and elasticity level. 14,58,59Elastometer equipped with highly sensitive noninvasive multiple adopter probe was used to measure the skin elasticity level just by placing the probe on the skin carefully.
The skin elasticity level increased continuously and gradually from day 1 to 90th day of study period by application of TSEG6 formulation, while the elasticity level is slightly and irregularly TA B L E 5 Kinetic modeling for TSEG6 and CEG drug release profile.

K 2 . 3 |
E Y W O R D S antiaging, ethosomes, stability, tocopherol succinate, transdermal permeation Preparation of tocopherol succinate-containing ethosomes (TSE) nanoparticles (NPs) TSE NPs (TSE vesicles) were prepared by modified quick cold method.Various TSEG formulations (N = 9) have been prepared , 3.45, and 34.76°C/min for TS (pure drug), physical mixture, and TS6F %EE = Total amount of drug − Amount of free drug × 100 Total amount of drug TA B L E 1 Composition of ethosomal vesicles (suspension).

3
rpm using CP41 spindle.The data were analyzed Rheocalc version 2.5.6.Ex vivo permeation study of TSEG6 and CEG was performed by using abdominal skin of Wistar rats through Franz diffusion cell.This study was performed for 24 h at 300 rpm stirring and temperature adjusted was 37°C.Receptor cell was filled with 1x PBS adjusted to pH 7.4 as a surrogated skin pH.The volume of the receptor cell was 12 mL in 1.76 cm 2 surface area.One milliliter samples were withdrawn at predetermined time intervals and the same volume was replaced with PBS at same sink conditions of pH and temperature.The collected samples were analyzed by UV-Vis spectrophotometry at λ = 285 nm.The drug diffusion rate of TSEG6 and CEG across the skin barrier was respectively determined by Fick's law of diffusion, using the following Equation (1): Where, F = Flux, Kp = Coefficient of apparent permeability, Ci = Initial drug concentration.Coefficient of apparent permeability (kp) was calculated as per the following Equation (2): where, Vr = volume of receptor compartment, Cr = concentration of drug in receptor compartment, A = area of opening of Franz diffusion cell, t = total time of permeation study (hours), Cd = concentration of drug in donor compartment.By steady state flux values of TSEG6 and CEG enhancement ratio (ER) was determined by the following Equation (3): according to the Declaration of Helsinki and after approval of the Ethical Committee Review Board of the University, The Islamia University of Bahawalpur, Pakistan.Adult women (N = 34), 22 to 35 years old) were enrolled as volunteers in this study after informed consent.Volunteers were selected by following Inclusion and exclusion by Declaration of Helsinki ethical criteria for human subjects used for medical research purposes.The volunteers less than 18 years old, smoking, pregnant, or with hypersensitivity to active ingredients were excluded.Volunteers with any skin diseases were not included in the study.Patients with any kind of disease was not included in study.The volunteers were free to quit the study at any time.Patch test used patch TSEG6 applied in the right arm and CEG applied in the left arm.Biophysical parameters, skin elasticity, moisture content, the presence of erythema, as well as melanin and sebum levels, were evaluated for 3 months two times at day night on clear washed skin.

2. 13 |
Statistical AnalysisTwo-way ANOVA, paired sample t-test, graph pad prism version 8.4.3 and SPSS statistic software version 20 WERE used for statistical analyses aiming at determining the changes and different effects produced by TSEG6 (optimized test formulation) and CEG (control formulation).Significant results were considered if p-value is less than 5% (p < 0.05).Results were measured as mean values ± standard error of means.Percent changes in various physicochemical parameters were calculated by the formula given below (Equation 4): where, A = specific time interval individual value, B = zero-hour value.

45 3. 7 |
to CH 3 stretch in TS formulation, PG, and phospholipid, respectively.The peaks at 1463.23 cm −1 , 1457.89 cm −1 , 1466.04 cm −1 are presented to CH 2 bend in TS formulation, PG, and PC, respectively.The peaks at 2854.02 cm −1 -2968.75cm −1 are assigned to C-H stretch.The peaks at 2866.60 cm −1 , 2925.13 cm −1 , 2874.29 cm −1 are attributed to C-H stretches of TS drug, PG, and phospholipid respectively.The peaks at 1066.00 cm −1 , 1064.42 cm −1 may represent C-O stretch in TS formulation and PC, respectively.TSEG6 and its excipients clearly shows no new peak formation and interaction.This confirms the stability of TSEG6.Disappearance or merging of new peaks in developed formulations spectra confirms the encapsulation of active drug agent and ingredients into the new vesicular formulation.14,31,Evaluation of ethosomal gels TSEG6 (test formulation) and CEG (control formulation) were evaluated comparatively for organoleptic changes in color, odor, appearance, feel after use, pH change (Figure5) and rheological behavior (Figure6), after placing both gels at various temperature storage conditions (e.g., 8°C, 25°C, 40°C and 40°C ± 75% RH) for 12 weeks (3 months).Carbopol-940, used as gelling agent, has high hydrophilic nature and good adhesive nature.Due to these properties, Carbopol increases the duration of contact time of applied formulation at the site of action, and can be used for ethosomal gel preparation.1,46,47 Comparison of pH analysis of A-Formulation test gel (TSEG6) and B-Control gel (CEG) after 12 weeks kept at 8°C, 25°C, F I G U R E 1 TEM micrograph of the selected tocopherol succinate-loaded ethosomes (TSEG6).Scale bar is mentioned.

F I G U R E 7
Average % change in melanin concentration after application of Tocopherol Succinate Ethosomal Gel (TSEG6, optimized test/drug loaded formulation) and control ethosomal gel (CEG, unloaded formulation) to volunteers for 12 weeks study measured by mexameter (N = 34).Significant overtime decreases in melanin content by TSEG6 compared to CEG (p < 0.01).F I G U R E 8 Average % change in erythema concentration after application of Tocopherol Succinate Ethosomal Gel (TSEG6, optimized test/drug loaded formulation) and control ethosomal gel (CEG, unloaded formulation) to volunteers for 12 weeks study measured by mexameter (N = 34).Significant overtime decreases in melanin content by TSEG6 compared to CEG (p < 0.01).F I G U R E 9 Average % change in skin moisture level after application of Tocopherol Succinate Ethosomal Gel (TSEG6, optimized test/drug loaded formulation) and control ethosomal gel (CEG, unloaded formulation) to volunteers for 12 weeks study measured by corneometer (N = 34).Significant overtime decreases in melanin content by TSEG6 compared to CEG (p < 0.001).
Comparison of pH values changes of TSEG6 and CEG at various temperatures stored at 8°C and 25°C, 40°C, and 40°C ± 75%RH for 3 months.
from Day 1 to 90th day of study period by application of TSEG6 formulation.While moisture level slightly increases but in irregular manner by application of control gel (CEG) and sometimes skin moisture level decreases by CEG.All volunteer's obtained results were measured as mean % changes (Figure9).When paired sample t-test applied to TSEG6 and CEG formulation significant resultsTA B L E 3