Formulation and characteristic evaluation of tacrolimus cubosomal gel for vitiligo

Abstract The present study investigates and highlights the potency of tacrolimus (TAC) for effective vitiligo management using cubosome gel formulation. TAC-loaded cubosome formulations were prepared by using the ultrasonication technique. The central composite design (CCD) statistical design was employed for optimization of TAC-loaded cubosome based on responsible variables like particle size and entrapment efficiency. The optimized (F-14) TAC-loaded cubosome was subjected to physicochemical characterizations, including, particle size and zeta potential analysis, SEM, drug loading and drug release profiling assessment. SEM imaging confirmed spherical morphology of TAC loaded cubosome. Drug release percentage from cubosome was found to be with controlled drug release of 73% for 12 hour and release kinetics follows zero-order and Fickian diffusion mechanism. The optimized TAC-loaded cubosome formulation was incorporated in Carbopol 934 for the topical application of TAC-loaded cubosome gel (TAC-CG) for vitiligo. Therapeutic efficacy of TAC-CG for effective control of inflammation in vitiligo condition was assessed using monobenzene (60%) induced model of vitiligo using C57BL/6 mice. TAC-CG treatment exhibited a significant reduction in the depigmentation as a response when compared to that of the monobenzene-sensitized normal control mice. Graphical Abstract


Introduction
Vitiligo is an acquired idiopathic dermatological disorder characterized by the selective loss of melanocytes, that is, hypomelanosis. [1,2]About 85-95% of the symmetrical lesion's cases represent non-segmental vitiligo whereas unilateral lesions commonly appear in segmental vitiligo.Younger people more prone to get segmental vitiligo when compared to non-segmental vitiligo. [3]Etiology of vitiligo is unknown, it may be due to lack of immunity power and reduction of melanocyte count in the skin , family history (heredity) or externals factors such as sunburn, stress or exposure to industrial hazards, [4] which results in the smooth, non-scaly, chalky-white macules found in several tissues in the skin, hair follicles, eyes, and inner ear. [5]The prevalence rate of vitiligo is about 0.5-1% of the world population.
The most common treatments for vitiligo are phototherapy, surgical ways, herbal drugs, red tones, yoga remedy, homeopathic and ayurvedic therapy and there are different topical formulations like steroids and immunomodulators betamethasone, valerate, triamcinolone, and very potent corticosteroids like alobetasol, fluticasone propionate, etc. [6] However, these treatments are not much effective, and the researchers are in advancing the basic understanding of the disease process and the treatment protocol over the years. [7]herefore, pharmaceutical technologies have encouraged the formulation scientists to explore alternative routes for the conventional route of drug delivery system. [8]The novel drug delivery systems extant an opportunity for formulation scientists to overcome many of these challenges by formulating [9] various novel drug delivery systems like sustained or controlleddrug delivery systems, novel carriers for controlled and targeted drug delivery, liposomes, niosomes, cubosomes, and so on, have been developed. [10]ubosomes are cubic-shaped self-assembled liquid crystalline nanoparticle.Glycerol monooleate (GMO) and phytantriol are the most common biocompatible lipids used for cubosome formation. [11]It is highly curved lipid bilayers that are surrounded by two identical, non-intersecting aqueous channels and exhibits interesting properties for applications as a drug carrier. [12]Cubosomes as nanocarriers prove to be of greater applicability in the delivery of lipophilic or poorly water-soluble drugs via the topical, ocular, or oral routes.
But the topical drug delivery systems must take several needs into account, ranging from ease of delivery to effectiveness of the drugs. [13]Hydrogel's formulations gain much attention from researchers due to their unique properties such as swelling in an aqueous medium, sensitivity to pH, temperature, or other stimuli. [14]Several polymers like cellulose derivatives, carbomers, natural gums, polyacrylates, and gelatin were commonly used in the preparation of hydrogel formulations. [15]Carbopol 940 is an acrylic macromolecule composed of acrylic acid repeating units.It is an ideal rheology modifier that can provide highly commendable viscosity and form sparkling clear hydro-alcoholic gels.It is extensively used in nanoproducts, particularly topical liquid, and circumfluous medications. [16]acrolimus is an immunomodulator of 23-membered naturally occurring macrolide lactone extracted from Streptomyces tsukubaensis. [17]This drug was first described by Tanaka and group in 1990.It acts on T cells and mast cells, inhibiting T cell activation and the production of proinflammatory cytokines, such as tumor necrosis factor (TNF), whose levels are higher in vitiligo lesion skin. [18]It prevents the release of proinflammatory mediators from mast cells by degranulation.The drug is used for treating various skin diseases such as vitiligo, psoriasis, atopic dermatitis and eczema, ulcerative colitis, dermatology, ophthalmology, and so on. [19]he present investigation aims to formulate a TACloaded cubosome nanoparticles (TAC-CG) for topical administration, to improve its solubility, permeability, and eventually its therapeutic efficacy in mono benzene (60%) induced vitiligo.

Experimental design
Cubosomes were prepared using a two-factor, five-level central composite design (CCD) to discover the most optimal amounts of variables.For each component, the amount of lipid (X1) and surfactant (X2) were used as independent variables (Table 1), and an experimental range was chosen.Particle size (Y 1 ) and EE (Y 2 ), which are physicochemical parameters of the generated cubosomes, were chosen as dependent variables (Table S1).A total of 13 trials were carried out, according to the CCD matrix prepared by Design-Expert software (Trial Version 11.0, Stat-Ease Inc., Minneapolis).

Preparation of cubosome dispersion
Cubosomes were prepared using an emulsification procedure that had previously been published.Poloxamer 407 (P 407) and lipid phase (GMO) was carefully weighed and melted in a hot water bath until a homogenous mixture was formed.At 70 � C, mechanical stirring was used to add 50 mg of tacrolimus to the molten liquid, followed by dropwise additions of distilled water.After adding all of the water, the dispersion was set aside for 24 hours to achieve equilibrium.Stirring brought about the development of a two-phase system, which was disrupted.To produce cubosome dispersion, the whole system was homogenized at 1500 rpm for 3 hours at room temperature.For subsequent research, the produced cubosome dispersion was maintained in glass vials at 4 ± 0.5 � C. [20]

Particle size, PDI, & zetapotential analysis
A particle analyzer (Zetasizer Nano ZS, Malvern Instruments, Malvern, UK) was used to assess the average particle size of the cubosome nanoparticles as well as their size distribution (polydispersity index; PDI) at a temperature of 25 � C. At a temperature of 25 � C, the zeta potential values of cubosome nanoparticles were also identified using laser Doppler anemometry. [21]3.2.Scanning electron microscopy SEM (Carl Zeiss NTS GmbH, SUPRA 40VP, Oberkochen, Germany) at 5.0 kV was used to assess surface morphology, and photomicrographs were produced at 40.0 kx magnification.[22]

Transmission electron microscopy
Cubosomes in a 5-ll solution were put in a 300-mesh carbon-coated copper grid and allowed to settle for 3-5 minutes.After that, the surplus fluid was wicked away with absorbent paper.The samples were then captured digitally using a Gatan axis-mount 2k x 2k digital camera and evaluated on a JEOL Model JEM 2100 120 kV transmission electron microscope. [23]

Entrapment efficiency
An ultracentrifuge tube was used to assess the entrapment efficiency (EE) of tacrolimus.Centrifugation at 3500 rpm for 15 minutes separated the free (unentrapped) drug from the entrapped drug in the cubosomes dispersion.UV-Vis detectors at 210 nm and 254.7 nm were used to determine the amount of unentrapped tacrolimus in the filtrate.The following equation was used to determine EE.
Dt and Df represent the quantity of total and free (unentrapped) drug in the sample, respectively. [24]

In-vitro release studies
The drug release investigation was conducted using a cellophane membrane.Tacrolimus-loaded cubosome dispersion was put to the dialysis membrane and submerged in 30 mL of 0.005% hydroxypropyl cellulose solution in distilled water with phosphoric acid to adjust the pH to 4.5.The samples were withdrawn at predetermined time intervals, that is, 0.5, 1, 2, 4, 6, 8, 10, and 12, hours and replaced with fresh dissolution media to maintain the sink condition.The drug concentration was determined by photometric observation of the samples at 210 nm.The above In-vitro release was adopted for TAC-CG formulation. [18]

Preparation of tacrolimus loaded cubosome based hydrogel
Cold mechanical gels of Tacrolimus cubosomal topical gels were made with Carbopol 940 as the gelling agent.Carbopol 940 in the necessary quantity was weighed.With constant stirring at 400-600 rpm, the weighed polymer was gently added to the beaker containing distilled water (40 ml).Tacrolimus loaded cubosome dispersion was added into above polymer solution and gently stirred for 1 hour.Propylene glycol, a penetration enhancer, was added while stirring.The gel's viscosity was then balanced by adding glycerol.As a preservative, methylparaben was used.With distilled water, the final quantity was increased to 100 gm.The created gel was stored for 24 hours to allow for full polymer desolvation, resulting in a white creamy hydrogel. [25]6.Determination of pH pH of formulations was determined by using digital pH meter by immersing the electrode in gel formulation and pH was measured.The measurement of pH of each formulation was done in triplicate and average values were calculated.The pH meter was calibrated with standard buffer solutions (pH 4 and 7).[26]

Viscosity
Viscosity of formulations was determined by using Brookfield (DV Pro-II) viscometer with small sample adaptor, spindle no.96 T. Speed was increased from 10 to 100 rpm and viscosity was noted in cps. [27]

Spreadability studies
The capacity of a cream or gel to uniformly spread over the skin, or the spreadability of semisolid formulations, plays a crucial role in the delivery of a standard dosage of a medicinal formulation to the skin and the efficacy of topical therapy.The spreadability and residence duration of the formulation at the application site can be affected by the formulation's flow characteristics.The formula S ¼ M L/t was used to calculate spreadability, where M is the weight (g) applied to the top slide, L is the diameter increase, and t is the time (sec). [28]

Drug content
The 1 gm of tacrolimus carbopol gel and tacrolimus-loaded cubogel were transferred to a 10 ml volumetric flask and diluted with ethanol. 1 cc of this solution was then diluted further with ethanol.A UV-visible spectrophotometer was used to determine the drug content by measuring the absorbance at 295 nm. [29]

Stability studies
TAC-loaded cubosomes and TAC-CG were checked under specified temperature and humidity conditions (25 � ±2 � C/ 60%RH ± 5%RH).Samples were collected at the end of the 90th day to determine the particle size, polydispersity index (PDI) and EE(%) for TAC-loaded cubosome gel and pH and predability for TAC-CG.

In-vivo vitiligo-induced study
Four-week-old female C57BL/6 mice used for the study were acclimatized for one week in animal housing and fed regular food.The Institutional Animal Ethical Committee approved the study protocol YU/IAEC/10/2021.The vitiligo on the rear neck portion of mice was induced by the topical application of monobenzone cream (60%) daily for four weeks.The animals were observed daily for pigmentation in the presence of a veterinary physician.The pigmentation has been investigated and classified in different intervals (0, 7, 14, 21, and 28th Day).The pigmentation was assessed visually and mark using scores from 0 to 5 based on the following remarks (0 ¼ no pigmentation; 1 ¼ first signs of pigmentation (freckles); 2 ¼ light; 3 ¼ medium; 4 ¼ dark; 5 ¼ white pigmentation).After induction of vitiligo, animals were divided into three groups with six animals each and treated for four weeks.Group-1 animals received a single daily dose of buffer solution served as control, Group-2 received tacrolimus cubosome gel be the test group, and group-3 treated with marketed formulation served as the positive control.The depigmentation's of animals were monitored daily until the last day of treatment. [30]

Histological examination
Perilesional skin tissue samples were collected from the control and treated animals and fixed in 10% neutral buffered formalin.The tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. [31]The histological sections of the epidermis were photographed using Olympus AX70 optical microscope (magnification of 20 and 40�).

Result and discussion
The study aimed to develop cubosome nano dispersion by using an optimum concentration of surfactant and lipid quantity to obtain the desired characteristics of the stable cubosomal formulation.Surfactant and lipid could be the critical material attributes for obtaining a highly desirable cubosome nano dispersion.Preliminary experimental studies were executed to find out the desired concentration range of surfactant and lipid to form a cubosome formulation.
The central composite design (CCD) is a second-order experimental design that is connected with a small number of trials.CCD was used to perform a systematic optimization to estimate the extended impacts of independent factors (Lipid (X1) and Poloxamer (X2)) on response variables such as particle size (Y1) and entrapment efficiency (Y2).
In this study, CCD was used as an optimization statistical model for determining the independent variables that can produce optimal responses, developing polynomial equations using experimental results, implementing appropriate statistical tests, and selecting the best fit model.
As per CCD, statistical model 13 experimental trials were given by Design Expert Software Version 11 (Stat-Ease Inc., USA).Table S1 depicts results obtained for all the drugloaded cubosome formulations (F1-F13) for the response factors particle size (nm) and EE (%).Cubosome dispersion appears milky white cubosomal dispersions with no phase separation or visible aggregates.The results obtained as polynomial equations, ANOVA statistical values and interaction between independent variables for all the response factors were represented as 3D graphs.

Effect of process variables on particle size
As per the CCD model 13 formulations were carried out and the particle size range were observed 178-495 nm.A liner model was selected to anlayse the particle size (p < 0.001) where was lack of fit non-significant.The polynomial equation explains the effect of interaction between two independent variables on the response variable.
The effects of lipid (X1) and poloxamer (X2) on particle size were shown to be significant using ANOVA (Table S2).The material variables determining particle size and size distribution were surfactant and lipid content.Increasing the poloxamer concentration from 0.75% to 1.5% resulted in a significant reduction in particle size (nm).This could be due to a reduction in interfacial tension between the cubosomal nanoparticles and the surrounding aqueous phase, resulting in the formation of smaller, non-aggregated nanoparticles. [32]Increased lipid concentration (0.4-0.8%) affects particle size directly proportionate to the amount of lipid utilized in formulation, as shown in Figure 1 as a 3D response graph.As previously indicated, increased concentrations of poloxamer 407 resulted in the formation of smaller particles with a lower volume for holding drugs.

Effect of process variables on entrapment efficiency
Table S1 indicates the EE percent of 13 produced formulations.The positive result in the quadratic equation indicates that lipid concentration (X1) had a substantial and favorable influence on entrapment efficiency (Y2).Table S3 shows that the ANOVA for percent EE (quadratic model) suggests that the model is significant for the response parameters chosen.The correlation coefficient (R 2 ) value of 0.9868 and the coefficient of variance percent of 3.65 for response factor percent EE are reported.
After fitting the data, Design Expert software was used to generate an appropriate polynomial equation for the response factor, which included both main and interaction variables.Increasing the lipid concentration boosted drug entrapment in the produced cubosomes, but increasing the poloxamer concentration had the opposite effect.The enhanced solubilization of tacrolimus by providing more capacity for tacrolimus to be added to its internal lipid structures might explain the direct relationship between EE percent and lipid content.The drop in EE percent with increasing poloxamer, on the other hand, might be due to faster solubilization and partitioning of tacrolimus into the aqueous phase during the transition from cubic gel to cubosomes.Furthermore, using more poloxamer results in the production of smaller cubosomes with a reduced space for storing the medication. [33]he high EE (%) of tacrolimus in lipid is attributed to its high lipophilicity, which encouraged its entrapment in the cubosomal internal structure. [34]he EE (%) of all 13 formulations were listed in table and it's found to be in the range of 78.5 ± 2.1 to 99.1 ± 3.8%.Observing results clearly indicate EE (%) of prepared formulation mainly influenced by lipid and surfactant concentration.3D response graph (Figure 2) illustrated an increase in the GMO concentration (0.4-0.8%) which enhances the entrapment efficiency.
Higher concentration of GMO could cause maximum solubility of hydrophobic drug (tacrolimus) occurs by providing more space to incorporate in the lipid matrix.Similarly, amount of surfactant changes (0.75-1.5%) in formulation, the entrapment efficiency decreased which may be due to a higher concentration poloxamer 407 tend to have solubilization and partitioning of the drug into the aqueous phase, during phase changes from cubic gel to cubosome dispersion.
To develop cubosomes with high entrapment effectiveness, it would be preferable to utilize a larger percentage of GMO (0.8%) and a smaller percentage of poloxamer 407, respectively (0.75%). [35]

Optimization and validation
Based on the graphical optimization and checkpoint formulation compositions, validation of the experimental design and polynomial equation optimization of the formulation were done.The optimized cubosome formulation (F14) containing lipid (0.4%) and surfactant (0.87%) was evaluated for particle size and EE.The predicted particle size (nm) % EE values obtained from the design expert software and the experimental values were concomitantly compared with experimental responses in Table 2. Optimized formulation (F-14) showed experimental % EE (96 ± 2.13%) and particle size 163.7 nm (Figure S1) whereas, the predicted drug release value % EE was found to be 87.07%and predicted particle size was found to be 175.2.In addition, the percentage prediction error calculated for all dependent variables was found to be < 5%.The minimal deviation between the predicted and experimental values indicates that the mathematical model is well-fitted and is significant.The result also signifies that the generated model is valid and robust. [36]

Zeta potential
The improved (F-14) formulation's average zeta potential was determined to be 2.9 0.5 Mv.Due to the use of poloxamer 407 (a nonionic stabilizer) and GMO, these data suggested that the cubosomes had a neutral zeta potential (amphoteric lipid).The existence of ionized lipid molecules might explain the partial negative charge.Poloxamer 407 would cover the surface of the cubic lipid structures, providing anti-aggregation stability.As a result, while having a neutral zeta potential, tacrolimus would be stable in the face of aggregation. [35]

Morphology of the cubosomes
Individual particles at a nanometric scale with cubic ultrastructure and no trace of big aggregates were seen using SEM and TEM, respectively (Figures 3 and 4), which might be attributed to electrostatic repulsion and steric stabilization.The development of cubic-shaped structures, cubosomes, was aided by the interaction of GMO and Poloxamer 407 in the presence of water at a regulated temperature via hydrogen bonding. [36]

Determination of pH
The pH of the formulations was found to be 6.5 ± 0.71 to 6.8 ± 0.25 for cubogel and 6.43 ± 0.83 for optimized gel.However, the normal skin pH is varied from 4 to 7. So, the formulations that have this range of pH, and they are accepted and would not fabricate any skin irritation.

Viscosity
The viscosity of prepared cubogels is displayed in Figure 5. Rheological properties are important factors in the gel formulation and application, as they influence the product physical form, appearance, texture, and flow behavior.It was found that all cubosome-loaded hydrogel exhibited shear thinning flow since the viscosity decreased with increasing shear rate.

Drug release
The In vitro drug release study of the optimized F-14 formulation was performed for 12 h in buffer media (7.4 pH simulated phosphate buffer) to assess the drug release profile and its mechanism.As shown in Figure 6, at the end of 12 hours, optimized F-14 formulation showed a biphasic release pattern over a period of 24 hours sustained drug release (% CDR at 12 hours 73.35%).
A burst release (10%) due to unentrapped tacrolimus was seen throughout the first hour.The burst phase followed a persistent release pattern of drug from the entrapped cubosomes.After 12 hours, there was a maximum of 73.35 ± 0.41% drug release.Although it was filled with new buffer at each time point to preserve the sink conditions, it does not replicate the infinite sink circumstances found in vivo.Although the tacrolimus was rather well entrapped in the cubosomes, it is not required to create formulations with extremely high entrapment efficiency, as reported by Radhakrishna Nithya et al.They claim that a mix of entrapped and free medicines might improve drug penetration across the skin (Figure 6). [35]

Release kinetics
The mechanism of drug release was explored by subjecting the data to kinetic analysis by fitting to various mathematical equations and models viz., zero order, first order, Higuchi and Peppas models.On the basis of higher regression values obtained, the F14 formulation followed the Korsmeyer-peppas model (R 2 ¼ 0.9923) of drug release with a release exponent value "n" of 0.74 indicating that the drug release data follows a non-Fickian diffusion mechanism of drug release with anomalous diffusion type in Table 3.

Gel drug content
Around 97% of the medicine is contained in the gel system in terms of content homogeneity.Normally, there was no medication loss in a gel system, although we observed a 3% deficiency.This can be related to the loss of formulation intermediates during handling, clinging to reaction vessels/ glassware, and before being transported into a gel system for final product formation.These findings showed that tacrolimus was uniformly mixed and distributed inside the created hydrogel and that it was preferred for topical formulation due to its more efficient controlled drug release. [37]

Spreadability
The hydrogel containing tacrolimus-loaded cubosomes had a high spreadability of 11.6 ± 0.11 g cm/sec.The proposed hydrogel formulation containing tacrolimus-loaded cubosomes may result in greater patient compliance due to its spreadability and ease of administration.Improved spreadability ensures that the gel will be spread more intimately with the skin, increasing the contact area for a small amount of the formulated gel and, therefore, delivering a more significant amount of medication due to the increased surface area for drug transposition through skin layers. [38]

Stability studies
Particle size and polydispersity index (PDI) of TAC loaded cubosome after the storage period of 3 months was 187.7 ± 13 nm and 0.310, respectively.EE of TAC loaded cubosome as 86.89%.Based on stability data, the nano formulation was found to be stable without aggregation even after under specified temperature and humidity conditions (25 � ± 2 � C/60% RH ± 5%RH) for the period of 3 months.
Similarly, after 3 months TAC-CG showed pH 6.5 and spreadability 11.90 ± 0.2 g cm/sec.Hence, it is clear that TAC loaded cubosome and TAC-CG are highly stable over a reasonable period of time at specified temperature and humidity.

In-vivo vitiligo study
Topical application of monobenzone (60%) resulted in depigmentation in mice.A minor pigmentation appeared initially in the applied area and gradually expanded.The white patches spread over time, with the possibility of substantial depigmentation over most parts of the body.At the end of four weeks, each animal applied with monobenzone showed a complete depigmentation and defoliation.Severe inflammation with a massive exudate was observed on the rear neck.A previous study revealed that the monobenzone (60%) application-induced depigmentation on mice supports our results. [30]The animals with depigmentation were treated with cubosome gel and marketed formulation and compared with the untreated control.Photographs of the neck portion after treatment are shown in Figure 7.The results showed a significant reduction in depigmentation in animals treated with cubosome gel and marketed formulation compared to the untreated animals.The animals treated with cubosome gel formulation commence repigmentation within 60 days.Patchy repigmentation was observed in the marketed formulation-treated group, whereas repigmentation is homogenous in the cubosome gel treated group.Observations for a couple of months after treatment revealed that the repigmentation process was significant and comparable in both cubosome gel and marketed formulation.Overall, cubosome gel may provide a substantial cure for vitiligo (Table S4).

Histopathology
Back skin samples were stained with hematoxylin-eosin and cell infiltrate were evaluated.Histological analysis and its representative pictures are shown in Figure 8.The melanogenesis of hair follicles in C57BL/6 mice has been recognized as a useful model for researching pigmentation in vivo.It is important to note that melanocytes are localized exclusively in the hair follicles in the mouse dorsal skin in contrast to humans. [39]Topical cubosome gel formulation treatment, as well as marketed formulation, showed noticeably enhancement of melanin inside of hair follicles.Treatment with cubosome gel noticeably increase the numbers of melanin-containing follicles, the basal melanocytes in the skin of mice.Compared with the untreated control group, the vitiligo group showed a significant decrease in melanin-containing follicles, basal melanocytes, and melanin-containing epidermal cells.The number of melanin containing hair follicles in the treated areas was found to be increased in the formulation-treated group.Although vitiligo is an acquired achromia of the skin, the primary cause is a deficiency in melanogenesis, and various researchers have concentrated on the histology of basal melanocytes in lesional parts of the skin.This has been demonstrated that biologically active melanocytes and basal melanocytes that exhibit tyrosinase activity when incubated with appropriate materials are rarely present in vitiligo lesional sites. [40]The developed formulation also raised the amount of melanincontaining epidermal cells, indicating that it could treat mice with vitiligo and can be used as a better alternative therapy.Through simultaneous melanocyte regeneration, proliferation, and migration, effective vitiligo therapy options should slow disease progression and improve repigmentation. [41]

Conclusion
Tacrolimus-loaded cubosome gel was successfully fabricated from submicron o/w cubosome dispersion incorporated using Carbapol 934.The optimized cubosome formulation (F-14) has a particle size of about 163.7 nm and entrapment efficiency of about 96.0%.TAC-loaded cubosome gel follows a controlled released profile and drug release kinetics follows zero-order kinetics.Therapeutic efficacy of TAC-CG for effective control of inflammation in vitiligo condition was assessed using monobenzene (60%) induced model of vitiligo using C57BL/6 mice.TAC-CG treatment exhibited a significant reduction in the depigmentation as a response when compared to that of the monobenzene-sensitized normal control mice.Therefore, it is concluded that TAC-CG would be suitable for topical administration owing to their ability to control its release and effective therapeutic management of vitiligo.This is the first pre-clinical report study for nanoparticle drug delivery-based tacrolimus-loaded cubosome gel for vitiligo by using monobenzone (60%) induced in C57BL/6 mice.A few pre-clinical research finding Zhu et al. and Moreira et al. [42,43] based on monobenzene-induced vitiligo model were observed in plant materials such as Pyrostegia Venusta and epigallocatechin-3-gallate (EGCG) for vitiligo treatment, respectively.
We observed numerous clinical studies on various drugs such as tacrolimus, and dexamethasone and their conventional formulations like tacrolimus ointment/cream formulations studies were conducted for the treatment of vitiligo.Our present attempt will explore performing many pre-clinical studies for the treatment of vitiligo by using a novel drug delivery system for better patient compliance.

Figure 4 .
Figure 4. Transmission electron microscopy of (A) TAC loaded cubosomes at 1 mm and (B) TAC loaded cubosomes at 2 mm.

Figure 8 .
Figure 8. Histopathology images: lymphocytes infiltrate the basal layer of the epidermis (arrow heads) in very low numbers at the junction of epidermis (B).Melanin containing macrophages are present in low numbers in the superficial dermis (arrow) and are accompanied by a very mild, mononuclear, perivascular, inflammatory cell infiltrate (B) and (C).The epidermal architecture is not significantly altered.Hematoxylin and eosin stain images in 40�.