Combinatorial drug‐loaded quality by design adapted transliposome gel formulation for dermal delivery: In vitro and dermatokinetic study

Ursolic acid is a powerful drug that possesses many therapeutic properties, such as hepatoprotection, immunomodulation, anti‐inflammatory, antidiabetic, antibacterial, antiviral, antiulcer, and anticancer activity. Centella asiatica (L.) Urban (Umbelliferae) contains a triterpene called asiatic acid, which has been used effectively in traditional Chinese and Indian medicine system for centuries. Anticancer, anti‐inflammatory, and neuroprotective properties are only some of the many pharmacological actions previously attributed to asiatic acid .


Materials and Methods:
The optimize transliposome for accentuated dermal delivery of dual drug.The optimization of drug-loaded transliposome was done using the "Box-Behnken design."The optimized formulation was characterized for vesicles size, entrapment efficiency (%), and in vitro drug release.Additionally, transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), and dermatokinetic study were performed for further evaluation of drug-loaded optimized transliposome formulation.
In vitro drug release of ursolic acid and asiatic acid transliposomes was found to be 85.12 ± 2.54% and 80.23 ± 3.23%, respectively, as compared to optimized ursolic acid and asiatic acid transliposome gel drug release that was 67.18 ± 2.85% and 60.28 ± 4.12%, respectively.The skin permeation study of ursolic and asiatic acid conventional formulation was only 32.48 ± 2.42%, compared with optimized combinatorial drug-loaded transliposome gel (79.83 ± 4.52%) at 12 h.After applying combinatorial drug-loaded transliposome gel, rhodamine was able to more easily cross rat

| INTRODUC TI ON
Worldwide, cancer is one of the leading causes of death for adults, based on estimates from the Surveillance and Health Equity Science, American Cancer Society, Atlanta, Georgia.As per recent literature survey, 1 918 030 fresh cancer cases were reported, and 609 360 cancer deaths are estimated to occur in the United States.The leading cause for death among cancer is lung cancer. 1 According to the Registrar General of India (2022), the projected cancer burden in India for 2021 was 26.7 million.Disability Adjusted Life Years adjusted mortality to incidence and is expected to increase to 29.8 million in 2025. 2 Triterpenoids, which have more than 20 000 members among the plant kingdom, are a significant family of natural compounds. 3,4ntacyclic triterpenes are a unique family of triterpenoid natural products that have distinct biological properties, as demonstrated in clinical and preclinical studies. 5,6As a hydroxy pentacyclic triterpene acid, ursolic acid (3b-hydroxy-12-urs-12-ene-28-oic acid) has been found extensively in medicinal plants and is a component of the waxy coatings present on a number of fruit species including apples, pears, olives, prunes, and cranberries.Due to its low water solubility and difficulties in permeating some biological membranes, the biopharmaceutical classification system (BCS) placed UA in class IV as a medication with minimal pharmacological activity.The low oral bioavailability of drugs in this class is due to both their slow disintegration and their limited gastrointestinal mucosa penetration. 7,8Ursolic acid (UA) is a powerful drug that possesses many therapeutic properties, such as hepatoprotection, 9,10 immunomodulation, 11 anti-inflammatory, 12,13 antidiabetic, 14 antibacterial, 15,16 antiviral, 17,18 antiulcer, 19 and anticancer activity. 20Past studies have demonstrated that UA has multifunctional anticancer properties. 20,21Despite promising safety and efficacy profiles in cancer treatment, UA's poor bioavailability, fast metabolism, and low solubility prevented it from fulfilling its therapeutic potential. 22aditional Chinese and Indian Ayurvedic medicine have long relied on asiatic acid (AA), a triterpene derived from Centella asiatica (L.) Urban (Umbelliferae).Previous research has shown that along with its antioxidant, anti-inflammatory, and neuroprotective actions, AA also has a wide range of other pharmacological benefits, [23][24][25][26] it is also effective in combating cancer.
Therefore, in the present study, an approach has been made to develop ursolic acid and asiatic acid-loaded transliposomes (UA and AA-TL) for dermal delivery.Asiatic acid is a powerful antioxidant that soothes UV-induced inflammation and scavenges damaging free radicals such as reactive oxygen species, nitric oxide, and protein carbonyl groups.In addition, it protects collagen and elastin fibers from glycation, preserving essential elasticity, firmness, and youthful skin whereas ursolic acid is known to prevent the development of cyclooxygenase and lipoxygenase, inflammatory enzymes that aggravate the skin.This TL delivery method combines the benefits of liposome and transferosome delivery into a single, innovative strategy.Increased drug solubility, stability, and permeation were observed in TL vesicles with edge activator due to their superior permeation ability and deposition properties. 27,28The formulation's residence time at the application site could be improved if TL were to be transformed into a gel with a higher viscosity.
The purpose of this research was to establish and optimize UA and AA-TL by the modification of independent factors including lipoid S100, cholesterol, and sodium cholate concentration.Box-Behnken analysis was used to determine how different factors impacted the three dependent variables of vesicle size, entrapment efficiency, and in vitro release (BBD).Additionally, the vesicle morphology, skin permeability and penetration characteristics, antioxidant activity, cytotoxicity, and dermatokinetic studies of the optimized UA and AA-TL formulation were analyzed.

| Materials
Ursolic acid, asiatic acid, cholesterol, and triethanolamine were bought from Sigma-Aldrich.Lipoid S100 was obtained from Lipoid GmbH.Sodium cholate was procured from Thomas Baker.Carbopol skin, as observed by confocal laser scanning microscopy, in comparison with when the rhodamine control solution was used.
Discussion: The UA_AA-TL gel formulation absorbed more ursolic acid and asiatic acid than the UA_AA-CF gel formulation, as per dermatokinetic study.Even after being incorporated into transliposome vesicles, the antioxidant effects of ursolic and asiatic acid were still detectable.In most cases, transliposomes vesicular systems generate depots in the skin's deeper layers and gradually release the medicine over time, allowing for fewer applications.

Conclusion:
In overall our studies, it may be concluded that developed dual drugloaded transliposomal formulation has great potential for effective topical drug delivery for skin cancer.

K E Y W O R D S
asiatic acid, in vitro release, translioposomes, ursolic acid, vesicular size 940 and polyethylene glycol 400 were procured from Merck.Other chemicals were procured from Merck.All solvents and chemicals used were of analytical grade, and HPLC water was used for all experiments.

| Preparation of combinatorial drug-loaded TL (CDLTL) formulation
To develop CDLTLs, methanol was combined with Lipoid S100, cholesterol, sodium cholate, and medicine (ursolic acid and asiatic acid) in a flask, and then, the solvent was removed using a rotary evaporator at a low temperature and pressure.The resulting lipid thin film was dried, precipitated, and rehydrated in a saline (pH 7.4) solution for 1 h while spinning at 150 rpm at room temperature.The resulting dispersions were then sonicated for 3 min in a probe sonicator to reduce their size to that of vesicles.The TLs were analyzed for their vesicle size, entrapment efficiency (in a percentage sense), in vitro drug release, and skin permeation (after being surgically removed).Three components and three layers of a Box-Behnken design were used, with some independent and dependent measures displayed in Table 1.

| Optimization of CDLTL formulation using Box-Behnken design (BBD) software
The 3-factor, 3-level BBD was applied for the optimization of formulation.As per the Design Expert® software version 13, 17 combinatorial drug-loaded TL formulations (Table 2) were obtained and evaluated.ANOVA provision offered in the software was employed to establish the statistical validation of the polynomial equations generated by Design Expert®.The independent responses selected were Lipoid S100 (X 1 ), Sodium cholate (X 2 ), and Cholesterol (X 3 ), and the dependent responses were vesicle size (Y 1 ), PDI (Y 2 ), and entrapment efficiency (Y 3 ) (Table 1).

| Vesicle size and polydispersity index (PDI) of optimized formulation
A zetasizer (Nano ZSP, Malvern) was used to estimate the vesicle size and PDI of the improved formulation at 25 ± 1°C.The formulations were diluted in saline (pH -7.4), and the results were measured three times.

| Morphological analysis by TEM
The improved TL formulation was analyzed morphologically using transmission electron microscopy (TEM; JEOL, JEM 1010).A diluted sample was dropped onto a copper grid, allowed to dry, and then dyed with phosphotungstic acid at a concentration of 1% (w/v). 29,30

| Entrapment efficiency (%EE)
Ultracentrifugation was used to assess the TL formulation's % EE.
For the purpose of isolating the free ursolic acid, two milliliters of the optimized-TL formulation were placed in a centrifuge (Cooling Centrifuge, C24, REMI Ins.Ltd.) tube and spun at 25 000 rpm for 1 h at 4°C.The concentrations of ursolic acid and asiatic acid were measured at 217 and 220 nm in diluted and filtered samples of the collected supernatant using ultraviolet (UV) spectroscopy.The following formula was used to determine the entrapment efficiency (in percentage terms) where %EE= Percentage entrapment efficiency.

| Evaluation of in vitro drug release
Using the dialysis bag diffusion method, we measured the drug release from CDLTLs gel and a combinatorial drug-loaded solution formulation.Dialysis bags were filled with the optimal formulations and then submerged in 50 mL of room temperature phosphate buffer (pH 7.4).At 0, 0.25, 0.5, 1, 2, 4, 6, 12, and 24 h, 1 mL samples were taken out and replaced with fresh dissolving media.The concentrations of ursolic acid and asiatic acid were measured in a UV spectrometer at 217 and 220 nm, respectively.Several mathematical models were used to approximate the in vitro drug release data. 31

| Antioxidant properties
At room temperature, the ursolic acid and asiatic acid in CDLTL's optimized formulation were tested for their antioxidant effects Spectroscopy at 517 nm was used to analyze the color shift.3.50 mL of 70% ethanol and 0.30 mL of DPPH solution made up the blank sample. 32Antioxidant activity as a percentage was used to characterize the sample's ability to neutralize free radicals. 33The formula used to determine the percentage of antioxidant activity is as follows: Abs.indicates Absorbance. 32

| Preparation of combinatorial drug-loaded Transliposome gel
The optimized transliposomes formulation was transformed into a gel formulation so that CDLTLs could rapidly detach from the skin.
Overnight, 100 mg of carbopol 934 was evenly distributed in 10 mL of double-distilled water (0.75% w/w) to allow full swelling of the carbopol.The pH was then adjusted with triethanolamine, and the dispersion was finished off with 15% w/w polyethylene glycol 400 and 0.1% chlorocresol (as a preservative).The final step in making a homogeneous gel formulation was to introduce the optimized UA and AA-TL dropwise into this preformed gel based while continuously stirring.Abbreviations: X 1 , Lipoid S100 (mg); X 2 , Sodium cholate (mg); X 3 , Cholesterol (mg); Y1, Vesicles size (nm); Y2, PDI; Y 3 , Entrapment efficient (%).

| Skin permeation study
Rat skin was taken for the study although there is different rate of absorption of drugs as compare to human skin but we can compute and correlate it with human skin permeation and absorption. 36anz diffusion cells having diffusion area 0.785 cm 2 of 10 mL volume of receptor were used to measure the rate of skin penetration.
Non-occlusively, 1 g of the optimized gel formulation was applied to a sample of freshly removed skin (thickness 0.95 mm) from a rat.
Throughout the experiment, the phosphate buffer pH 7.4 receiver vehicle was regularly swirled (600 rpm) at 37 ± 1°C.At 0, 0.25, 0.5, 1, 2, 4, 6, and 12 h, 1 mL samples were taken from the receiver compartment through the sampling port and replaced with fresh vehicle right away.UV spectrophotometry was employed to ascertain the drug concentration.

| Skin uptake study by Confocal method
Confocal laser scanning microscopy was utilized to ascertain the formulation's skin penetration depth (CLSM).Extracted rat skin was placed on two distinct Franz diffusion cells and incubated for 8 h at 32 ± 2°C with either the optimal gel formulation loaded with rhodamine B or rhodamine B in hydro-alcoholic solution.Small portions of rat skin were dissected and mounted on glass slides with the stratum corneum facing upwards for examination using a confocal laser scanning microscope (Leica TC SPE-IIw, DMI 4000 RGBV Leica Microsystems).The rat skin was scanned optically using the confocal microscope's z-axis at 5 μm intervals.Fluorescence emission was optically excited at 488 nm with an argon laser and detected at 532 nm. 37

| Dermatokinetic study
In the in vitro skin permeation investigation, the CDLTL gel formulation was applied to rat skin mounted in Franz diffusion cells to measure the drug content in various layers of skin at various times.
However, in this experiment, we took the extreme measure of removing the Franz diffusion cell's entire skin at varying time points (0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, and 8 h). 38We used saline (pH 7.4) to remove any remaining formulation then after soaking the skin in warm water at 60°C for 2-3 min, the epidermis and dermis were removed from the skin sample by forceps.The removed skin layers were then chopped into small pieces and soaked in 5 mL of methanol for 24 h to extract UA and AA.After collecting the methanolic solution, UV spectroscopy was used to measure the quantity of ursolic acid in the filtrate.Marketed formulation, CDLTL gel, and AA gel content per cm 2 of skin vs time were graphed for both the epidermis and dermis separately.Dermatokinetic parameters T skinmax , C skinmax , AUC 0-8h , and K e were measured.PDI (Y 2 ), and entrapment efficiency (X 3 ) were chosen as dependent responses, while lipoid S100 (X 1 ), sodium cholate (X 2 ), and cholesterol (X 3 ) were chosen as independent responses (Y 3 ).Table 2 dis

| Response-1 (Y 1 ): effect of independent variables on vesicle size
Vesicle sizes varied from 85.36 to 134.13 nm throughout the 17 runs, with an average of 111.53 nm (Table 2).
According to equation ( 1), both lipoid s100 and cholesterol positively influenced vesicle size as well.Because lipoid s100 and cholesterol both expand the bilayer, increasing their amounts causes the vesicle size to increase.
Increases in sodium cholate concentration from 5 mg to 15 mg resulted in a decrease in the size of the ursolic and asiatic acid-TL vesicles, indicating that sodium cholate is also required for vesicle production.

| Response-2 (Y 2 ): effect of independent variables on PDI
The PDI value from all 17 formulations ranged from 0.230 to 0.262 with an average value of 0.2422 (Table 2).
The equation ( 2) indicated that sodium cholate produced a negative effect on PDI.When the concentration of sodium cholate was increased from 5 to 15 mg, the PDI of ursolic and asiatic acid decreased. ( Vesicle size = +85.95− 0.5487 (2) However, both lipoid S100 and cholesterol had a beneficial effect on PDI.The PDI value rose when the cholesterol concentration was raised from 5 mg to 15 mg.
Entrapment efficiency was significantly influenced by Lipoid S100 and sodium cholate in equation ( 3).We found that the effectiveness with which ursolic acid and asiatic acid were entrapped in TL vesicles correlated positively with the amounts of lipoid S100 and sodium cholate present.Sodium cholate concentrations between 5 and 15 mg marginally improve entrapment efficiency.As the number of TL vesicles increases, the domain's dimensions grow, and more room is available for the trapping of drug molecules. 39cause of its ability to prevent leakage, stabilize bilayers, and slow the permeation of solutes present in the aqueous core of the vesicles, cholesterol was incorporated into the formulation for the purpose of producing stable vesicles. 29Cholesterol has a detri- before.When cholesterol levels go too high, it can cause the vesicular membranes to lose their normal bilayer shape and leak their contents, which can be harmful to the patient. 40 achieve maximum effectiveness, the ursolic and asiatic acid-TL formulation was optimized using the point prediction approach included in the BBD program.Here, we show that UA_AA-TLs with lipoid S100 (100 mg), cholesterol (10 mg), and sodium cholate (10 mg) met the criteria for a well-balanced formulation.

| Morphological study of CDLTL
Optimized CDLTL formulation TEM analysis confirmed the wellidentified sealed structure of the produced vesicles, which were found to be spherical in shape and uniform in size (Figure 3B).Comparable results were obtained when the vesicle size was calculated using the dynamic light scattering method and a Zetasizer device, as shown in the aforementioned figure (Figure 3A).The vesicular structure of ursolic acid and asiatic acid was clearly visible in the TEM micrograph, and there was no evidence of any crystalline substance.

| Evaluation of in vitro drug release
Drug release was 85.12 ± 2.54% from UA-TL and 80.23 ± 3.23% from AA-TL in the optimized CDLTL and CDLTLG formulation at 37 ± 0.5°C under continuous stirring at 100 RPM using the dialysis bag method; in comparison, the drug release percentages from the optimized UA-TL gel and the optimized AA-TL gel were 67.18 ± 2.85% and 60.28 ± 4.12%, respectively (Figure 4).Controlled release of the entrapped drug over a period of 24 h is demonstrated by our method of administering ursolic acid and asiatic Acid using this TL gel platform.However, we observed that the created UA and AA-TL showed that the release of these compounds followed a fickian diffusion with an R 2 value of 0.9912 and a n value of 0.17.

| Antioxidant properties
The antioxidant capacity of UA and AA has been well demonstrated, with studies demonstrating the agent's effectiveness in scavenging free radicals and protecting against lipid peroxidation.The antioxidant power of the improved UA and AA-TL formulation was measured in comparison with that of a conventional ascorbic acid solution.
Antioxidant activity in ascorbic acid solution was 94.32%, whereas in the UA and AA-TL optimized formulation, it was only 73.26%.This finding substantiates the formulation of ursolic acid and asiatic acid-TL gel's antioxidant properties.It was determined that UA and AA's antioxidant power was not affected by their integration into the TL gel.In addition, the antioxidant power of UA and AA is maintained, leading to an uptick in catalase, superoxide dismutase activity, and glutathione peroxidase within cells.

| Estimation of pH and the analysis of texture of the optimized UA-TL gel formulation
When developing a transdermal formulation, the pH of the solution is a crucial factor to consider, especially when the topical delivery system is meant to treat cancer.Healthy cells and their surrounding extracellular environment have a pH of 7.2.
Accordingly, the pH value of 6.5 observed in the UA and AA-TL gel formulation was well within the appropriate limits for topical administration to skin.Based on the results of the texture analysis depicted in Figure 5, the UA and AA-TL gel had a firmness of 221.65 g, a consistency of 220.38 g.s, a cohesiveness of −145.58 g, and a viscosity index of −56.10 g.s.The results suggest that the UA and AA-TL gel formulation has a consistent look and feel, with no lumps or uneven spots.

| Skin permeation study
Cumulative ursolic acid permeation from the UA and AA-CF was only 32.48 ± 2.42%, compared with up to 79.83 ± 4.52% from the optimized CDLTL gel at 12 h (Figure 6).The squeezing of the elastic nature of TLs through the stratum corneum, where edge activators enable such permeation, could account for this notable increase in penetration through the rat skin.Due to the development of hydrotaxis, which is governed by the laws of elastomechanics, the difference in transpore hydrostatic force between the two sides of skin is responsible for this penetration.The fluidity of the membrane is changed reversibly by the elasticity of the vesicular structure to enable the passage of the vesicles through the pores. 28

| Skin uptake study by Confocal method
Confocal laser scanning microscopy revealed that when rhodamine B was incorporated into the optimized CDLTL gel, the gel was well TA B L E 3 Dermatokinetic parameters of market formulation, UA and AA-TL, UA gel and AA gel.

| Dermatokinetic study
The statistical analysis using one factor ANOVA is shown in Table 3, and it shows the relative content of ursolic acid and asiatic acid in layers of rat skin's dermis and epidermis following treatment with commercial formulations of UA and AA-TL, UA gel, and AA gel formulation at various time intervals denoted in Figure 8.When comparing the quantities of ursolic acid and asiatic acid in the epidermis and dermis of rat skin treated with market formulation, UA and AA-TL, UA gel, and AA gel, the ursolic acid and asiatic acid-TL gel produced significantly greater concentrations of ursolic acid and asiatic acid as measured both C skin max and AUC 0-8 (Table 3).Perhaps, the ability of vesicles to enhance partitioning through skin lipid bilayers is responsible for the maximal retention of UA and AA-TL gel.
The T Skinmax of the ursolic acid and asiatic acid-TL gel was like that of the UA gel and AA gel in the epidermis.The results show that after 30 min of skin application, UA and AA concentrations are detectable.After topical administration, the C max was reached in the dermis and epidermis within 1.5 and 2 h with the UA and AA-TL delivery, respectively, which was significantly faster than with the commercial formulation.Results showed that UA and AA concentrations declined with time until 8 h, at which point they were detectable.
Based on the results of the skin permeation study and the confocal laser microscopic study, where the increased penetration of the formulation was evident due to the elasticity of the formulation and F I G U R E 8 Shows BRC concentration on (A) Epidermis and (B) dermis after topical application of market formulation, UA and AA-TL, UA gel and AA gel on excised rat skin.

using the 2 , 2 - 100 TA B L E 1 Y 1 =
diphenyl-1-picryl-hydrazyl (DPPH) technique.The % EE = (Total UA _ AA − UA _ AA in supernatant) Total UA _ AA × Independent variables with their levels and dependent variables with their constraints in Box-Behnken design for the preparation of CDLTLs.Vesicle's size (nm) Minimize Y 2 = PDI Minimize Y 3 = Entrapment efficient (%) Maximize violet color of DPPH free radical solution at room temperature disappears when it reacts with an antioxidant.Gels containing 0.5 mL of UA and AA-TL were dissolved in 3 mL of alcohol, combined with 0.3 mL of DPPH ethanolic solution, and left in the dark for 100 min.

3 | 3 . 1 |
RE SULTS AND D ISCUSS I ON Optimization of developed formulation by Box-Behnken design (BBD)When it came to formula development, the BBD software planned out 17 iterations over three focal points.The results of all 17 tests indicated that the quadratic model best fit the data.Vesicle size (Y 1 ), plays the R 2 , standard deviation, and percentage CV values for the three replies.The 3D graph shows how the size, PDI, and entrapment efficiency of vesicles change as a function of the chosen independent factors (Figure 1).In Figure 2, we can see a quantitative comparison between the experimental values and the projected values of the responses.
mental impact on entrapment efficiency, as shown in the preceding equation.The entrapment efficiency of the ursolic and asiatic acid-TL decreased as the cholesterol concentration climbed from 10 to 20 mg.The new numbers line up with what has been reported

E 1
Representation of 3D surface plot on the effect of independent variables on (A) Vesicle size, (B) PDI and (C) Entrapment efficiency.(85.95 nm), PDI value (0.232), and entrapment efficiency (87.12%), the values obtained by the Design Expert software were quite near to the projected values.An extensive battery of tests, including a dermatokinetics analysis and assessments of vesicle morphology, in vitro drug release, antioxidant activity, pH, texture, skin permeability, and depth of penetration, were conducted on the optimized CDLTL formulation.

F I G U R E 2
(A-F) the linear correlation plots (A, C, E) between actual predicted vs actual values and corresponding residual plots (B, D, F) for responses vesicle size and entrapment efficiency and in vitro release of UA_AA-TL.
gel formulation was preferable for delivering UA and AA, as the release pattern of UA and AA from the UA and AA-TL was significantly high in an aqueous environment within the constraints of our experimental design.A number of models, including the zero order, First order, Higuchi, and Korsmeyer-Peppas model, were applied to the data acquired from the in vitro release investigation.When deciding the order of release, the greatest correlation coefficient (R 2 ) value was favored.Optimized UA and AA-TL gel showed the highest R 2 value for the Higuchi model (R 2 = 0.9642), followed by the first order (R 2 = 0.952) and zero order (R 2 = 0.8306) models.The best fit model was discovered to be Higuchi's, and it was determined that the optimal UA and AA-TL gave the maximum value of the correlation coefficient.The Korsmeyer-Peppas model was used to analyze the UA and AA release from the optimized UA and AA-TL, and the results

F I G U R E 3 F I G U R E 7
(A) Average vesicle size using zetasizer, (B) Transmission electron micrograph of optimized UA_AA-TLs formulation.F I G U R E 4 In vitro drug release from UA_AA-TL and UA_AA gel formulation at pH 7.4.F I G U R E 5 Texture analysis diagram of optimized CDLTL gel formulation.F I G U R E 6 Ex vivo graphs show cumulative amount of UA_AA permeated through rat skin using UA_AA-TL gel and UA_AA-CF gel.Confocal laser microscopy of Rhodamine B loaded TL (A) and Rhodamine B solution (B) showing a depth of penetration 49.9 μm and 25.0 μm across rat skin.
absorbed and showed deeper penetration up to a depth of 49.9 μm (Figure7A), while the rhodamine B in the hydro-alcoholic solution was retained in the top layers of skin (permeating only to a depth of 25 μm, Figure7B).Maximum fluorescence intensity was observed in the dermal midsection, suggesting deeper penetration of the gel formulation into the lower epidermis after initially penetrating only the uppermost layers of the skin.The produced CDLTL gel was found to be effective at carrying rhodamine B dye into the dermal layers of rat skin.One possible explanation is that the UA and AA-TL are able to penetrate animal skin because of flaws in the lipid packing of the subcutaneous layer.When given transdermally, the encapsulated medication is transported to the subcutaneous tissue inside of these vesicular lipid particles.28

34,35 2.10 | Estimation of pH and texture of the CDLTL gel formulation
Observed responses in BBD software for the optimization of UA_AA-TL formulation and summary of results of regression analysis for responses Y 1 , Y 2 and Y 3 for fitting to quadratic model.
28 a rate of 2 mm/s on two separate occasions, with a 20-s delay between the end of the first compression and the start of the second.The gel's mechanical properties, including its firmness, consistency, cohesiveness, and index of viscosity, were mapped out on a texture analysis curve.28%antioxidantactivity = (Abs. of blank − Abs. of the sample) Abs. of blank × 100.TA B L E 2