DESIGNING OF NOVEL TOPICAL IN SITU POLYMERIC FILM-FORMING SOLUTION SPRAY FORMULATION OF ANTIFUNGAL AGENT: IN VITRO ACTIVITY AND IN VIVO CHARACTERIZATION

Objective: Voriconazole (VCZ) is a broad-spectrum antifungal medication that works by inhibiting fungal Cytochrome P450, preventing fungi growth. The current study aims at developing and characterizing an antifungal in situ film-forming polymeric solution spray containing VCZ for use in topical drug delivery systems. Methods: Optimized VCZ in situ polymeric film formulation was evaluated for Fourier transform infrared spectroscopy (FTIR), differential scanning calorimeter (DSC), X-ray diffractometry (XRD), Scanning electron microscope (SEM), in vitro and in vivo , ex-vivo investigation using abdominal rat skin and stability studies. The in vivo antifungal activity of the advanced in situ film was examined in albino Wistar rats. Results: The optimized batch contained 22% Eudragit RS 100 (ERS) and 4% Sorbitol. Based on FTIR, XRD, SEM, and rheological studies. Formulation ingredients of VCZ loaded topical in situ polymeric film spray were observed to be compatible and showed no evidence of precipitation, deformation, or discoloration. Diffusion test ( in vitro %), and ex-vivo drug diffusion % obtained 99.22%, and 97.45% respectively. The maximum inhibition zone was measured at 13±0.07 mm. The Wistar rat was employed as an animal model for skin irritation and antifungal studies. A study of short-term stability observed no significant modifications in the physical properties. Conclusion: The findings of the optimized VCZ topical in situ polymeric film spray formulation were satisfactory, demonstrating comparable improvement in superficial antifungal treatment. Voriconazole, Candida Albicans, In vitro and In vivo antifungal activity


INTRODUCTION
The fungal kingdom is unbelievably complex, and its representatives can extremely exist in many niches. Astonishingly, despite this outstanding versatility, relatively few human pathogens are found regularly. However, the human immune system can evade fungal infections. As a result, fungi have been considered harmless and seldom designated to be human fungal pathogens until the last two centuries. While some fungi are prime pathogens that cause human disease without a recognized deficiency, the second category of fungi is opportunistic pathogens [1,2].
Polymeric films have proven to be highly effective in topical therapy because they are easy to apply to the skin's surface and retain the medicine for a more extended time by resisting any modification, enhanced cosmetic appearance, including rubbing and washing or wearing clothes [3,4]. Polymeric films aid in the avoidance of difficulties or complications with systematically dosed forms and a semi-solid topical formulation. The most identifiable and easily appropriate topical procedure is evolving polymeric film because they produce the desired prolonged outcome while being conveniently and easily topical efficient. In situ polymeric film solution is a novel concept that may evolve unconventional dose methods applied to the skin, such as ointments, creams, gels, patches, and sprays [5][6][7].
Polymer film solution in situ spray is used in the form of liquid which, after solvent evaporated from their formulation, formed transparent in situ films that adhere to the skin within a very few seconds [8,9]. This formulation has four components: an active drug (VCZ), a second-generation triazole (which is used as a model drug), and the polymer (ERS) (which is capable of forming a clear and transparent film at skin temperature upon application), plasticizer as (Sorbitol), and absolute ethanol. However, 3 2 factorial design of the experiment (DoE) is a designed and structured technique; it can be used to determine the relationship between the effect of change in the concentration of the independent variables and its impact on the formulation by establishing a mathematical model [10,11].
This research aims at designing and characterizing antifungal in situ film-forming polymeric solution spray containing VCZ for topical drug delivery. This study mainly focuses on a wider variety of (In vitro, in vivo and Ex-vivo studies), the kinetics of drug release, skin irritation study, antifungal activity, and stability. The study of stability was carried out for six months in our previous study [12], which discussed and analyzed the essential factors, i.e., (solubility for drug and polymer, DSC, FTIR, water vapor permeability (WVP), moister absorption, moisture content, mechanical film properties, spray angle, and spray pattern). Finally, these polymeric filmforming solution sprays F-5 had advantageous characteristics, which could effectively be used as a base method for optimized VCZ in situ film to treat the fungal infections topical drug delivery.

Materials
The required chemicals products have been involved: Voriconazole (Yarrow-chem products Mumbai, India), Eudragit RS100 (ERS) (Vikram Thermo India Ltd Mumbai), Sorbitol (Loba Chemicals laboratory Mumbai), Ethanol (Merck life science PVT. Ltd), and other fine chemicals were purchased locally and from the pharmaceutical laboratory.

Preparation of polymeric solution in situ film
The preparation of Polymeric films has been prepared by dissolving various concentrations of (ERS), ranging from 20 to 24 % in absolute ethanol without plasticizer, and kept for overnight stirring continuously until a clear solution appears, which is followed by the addition of plasticizer with different concentrations, Sorbitol ranging from 2 to 4%. The weighed drug was precisely dissolved in 20 ml of the polymeric film solution with constant stirring at 1% w/v concentration in all the formulations [13,14], as shown in table 1.

Design of experiment (DoE) to optimize the formulation
A complete 3 2 design factory was used to optimize formulated in situ polymeric films. The polymer (X1 Eudragit RS100) and plasticizer (X2 sorbitol) concentrations were chosen as independent variables based on the existing related work. In contrast, (Y1) viscosity and (Y2) DT% as dependent variables were selected and VCZ was a constant value [15,16]. A statistical model is concerned with compositional response (degradation) Equation (1).
Where (Y) is the interactive response and (b0) is the arithmetical uncaring overreaction of 9 trails. The above interaction in the equation (Y) is the measurable result of preparation ingredients or various autonomous changes X and Y; b0 is the average arithmetic interaction; b1, b2, and b3 are the correlated coefficients of the elements X and Y. Factorial design details are provided in table 2. Variables representing each factor were included with an excipient each, with the corresponding qualitative levels. DESIGN EXPERT ® (version 10) software brought the optimized formulation.

Dry time
The solution was sprayed on the skin surface and glass slides, allowed to dry, and time (S) was recorded by a digitalized clock. The solution can be allowed to dry in a normal environment, and as a result, the film was formed [17].

Viscosity
The solutions were measured with a Viscometer of Brookfield (Brookfield-engineering lab. Inc., USA.) at a viscosity (25±1 °C). 20 ml of the sample was collected in the ULA cylinder, and the ULA S00 spindle was rotated at 10 rpm. The torque readings were much higher than 10%. Three measurements were taken, and the average was determined [17].
Where, AL-the quantity of the sample given at each distribution, Wtafter distribution of formula weight, W0-initial formulation weight before distribution, and Dn-density.

FTIR spectral study of the drug in situ film
The polymeric film was crushed using pestle and mortar until powder form was obtained and mixed with Potassium Bromide (KBr) in a 1:5 ratio (Powdered Polymeric film: KBr). With a holder's help, the mixture was pressed to form pellets and placed under an IR beam. Using KBr pellets, the spectra were read using a wavelength ranging from 400 cm -1 to 4000 cm -1 [18,19].

Differentials scanning calorimetry
DSC tests were conducted for optimized in situ film formulation using Shimadzu, DSC 60 apparatuses. The polymeric film was grinded in mortar-pestle until a fine powder was formed, then a required less quantity was transferred into sealed aluminum metal pans and scanned at 10 °C/min within a range of temperature of 20 °C-300 °C, and DSC thermogram was recorded. The liquid nitrogen was used as a coolant [20,21].

Scanning electron microscopy
The microstructure of the films was determined using a scanning electron microscope (JEOL JSM-IT300, Japan). Film samples have been stored for two weeks in an airtight desiccator containing silica gel. Then films were snapped in liquid nitrogen gently and at random smashed to examine the sample's cross-section [23,24]. Sample film and formulation as a solution was set up on copper stubs, gold-coated, and noted utilizing a step-up voltage of 10 kV.

X-ray diffractometry (XRD)
The XRD analysis of optimized film dried in situ polymeric formulation, pure VCZ, polymer, and the physical mixture was performed at room temperature, and the patterns were obtained using a Rigaku diffractometer. The patterns of diffraction were obtained by Ni-filtered Cu Kα radiation, where λ = 1.498 °A, under 20 mA, 40 kV voltage operation [22]. The samples were screened at 10 °-90 ° 2θ ranges, with experimental parameters set as scan step size of 0.02 ° for 2 seconds with a scan speed of 0.01 °/S.

Rheological study
The rheological performance of optimized polymeric in situ film and placebo formulation solution requires using the MCR100 controlled stress rheometer, having a coaxial cylinder with a radii ratio of 1.08477. The rheometer had a temperature-controlled unit for maintaining the temperature with a precision of 0.01 °C and a flowing water jacket was a part of the assembly used. The rheological factor, shear stress (Pa) was assessed straightly, rising to a shear rate of 250 s-1 with a 20-S interval and 25 shear stresses. US200 (Paar-Physica, Anton-Paar Gmbh, Austria) software was used to determine the shear rate. The rheological measurements at three different temperatures levels (4 °C, 25 °C, and 37 °C) were carried out corresponding to the storage area and the body temperature, respectively. All experiments were performed in triplicate, with a new sample taken for each assessment [25].

In vitro release study using cellophane membrane
In vitro diffusion study for the optimized VCZ in situ film solution spray was carried out by a cellophane membrane using Franz diffusion cell. The cellophane membrane was activated by soaking overnight in a phosphate buffer of pH 6.8. The membrane was kept between the donor and receptor compartment. In the donor compartment, 1 ml of polymeric solution was transferred. The receptor compartment had 20 ml of phosphate buffer and the entire surface of the membrane was in contact with it. The sample was withdrawn from the receptor compartment at intervals of 0, 15, 30, 60, 120, 180, 240, 300, 360, and 420 min. An equal volume of fresh phosphate buffer was added after sample collection each time. The amount of drug diffused was measured by UV spectroscopy at 256 nm [26,27].

Ex vivo drug diffusion studies
Franz diffusion methods {an area of 3 cm 2 } and receptor volume (25 ml) obtained from the skin (abdominal region) of the albino rat were used to study the skin permeation. The Rats were euthanized under anaesthesia, and the hair from the skin was detached entirely. Surgically, the skin from the abdominal piece of the rat was detached multiple times and it was cleaned with saline solution. Rat's skin was freed from the fat and subcutaneous tissue. The excised skin was fitted between two pumps of the Franz diffusion unit on both sides connected with the receptor intermediate and the SC side fronting towards the giver compartment; the optimized formulation weighing 0.1g was spread uniformly on the surface skin. The lower compartment of the receptor holds the phosphate buffer {pH 7.4}. Dimethyl Sulfoxide {2% v/v} was constantly stimulated with a magnetic agitator and was kept at 37±1 °C for a temperature of skins.
After the withdrawal of the samples, they were subsequently replaced with the similar phosphate buffer medium via sampling port of the diffusion unit at static time intervals of 0.5,1, 2, 4, 6, and 7 h. Withdrawn samples were analyzed by UV spectrophotometry [28][29][30][31].

Kinetics of drug release
The ex vivo research based on data on drug released were assembled in many testing types, including zero order, first order, Higuchi's type, and Korsmeyer-Pappas, to assess the kinetics of releasing optimized VCZ in situ polymeric film formulation. The following equations detail the function for each model calculation [32]. Zero-order: Where: K0 is the constant rate of zero-order in concentration units/time, t is time in minutes graph of concentration unit inconstant to time may produce a vertical line, the origin of the axes is intercepted at a slope equal to K0.

Organism and inoculums
The evaluation of in vitro antifungal activity of developed film forming spray based in situ film was done against Candida albicans implying agar diffusion method through well diffusion technique. Subcultures of Candida albicans ATCC 90028 were procured from June Enterprises PVT. LTD Navi Mumbai, India.

Well diffusion method
The configuration of Sabouraud's dextrose agar was examined in 250 ml of a pointed conical flask and was melted in 100 ml of purified water, and the pH was attuned to 5.6 to be sterilized in a hot air oven at 160 °C aimed, at a time of 60 min. After the accomplishment of sterilization, the medium was put aside at a chamber temperature of 0.5 ml watery suspension culture in NaCl 0.9 % to 5 × 10 5 CFU/ml were added to 100 ml of a medium at 47±2 °C and employed as a vaccinated layer. The average (20 ml) was transferred into a sterilized Petri dish to get 3-4 mm depth. It was confirmed that the medium layer is unchanging in thickness by employing a Petri dish on a smoothed surface. After settling down the medium of chamber temperature, with the assistance of a disinfected cork borer, cups of each 4 mm diameter were pierced and taken out from the Petri dish [33]. Sterilized solutions of placebo film (A), negative formulation (B), an optimized topical formulation based in situ film (C) in DMSO were added in respectively marked cups {0.1 ml of 0.1% w/v}. The solvent (DMSO) was employed as a negative control, and the whole study procedures were accomplished in sterilized conditions. The Petri dish was at that time nurtured for 24 h at 37 °C. After incubation, the zone of inhibition was evaluated in triplicate.

In vivo studies
In vivo research work are vital elements of current formulation and contemporary studies since they supply valuable evidence of effectiveness. Twenty-four adult female albino wistar rats weighing 200 to 250 g were used in this study. Ethical clearance was received, and all experimental animal protocols were endorsed by the Institutional Animal Ethics Committee (IAEC), JSS College of Pharmacy, Mysuru (Proposal no. 33/2019). The experiments were done according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA). These animals were kept free for a regular diet and water. Before the commencement of the investigation, all rats were acclimatized in wire cages for 12h light-dark cycle for seven days to allow them to adapt to the new environment [34].

Skin irritation studies
Irritation of skin study was done and carried out as per the Organisation for Economic Co-operation and Development (OECD) guidelines, and the study procedures were approved by the IAEC. The current research objectives were to examine any possible irritation occurrences of the topical application of polymeric film on rat skin. The hair on the dorsal side was shaved using an electric clipper and cleaned with alcohol for 4 h before starting the study [35]. Ten healthy female Wistar albino rats (200-250 g). Divided into three groups as (n=5): 1 st Group G1: Placebo polymeric in situ film, 2 nd Group G2: Optimized VCZ polymeric in situ film, and 3 rd Group G3: Negative control.
Formulations were topically applied to the target skin area (0.25 ml/kg/d). It is used by (1 ml) plastic syringe. After the application, careful clinical observation was made for any clinical signs of itching or redness, dryness, papule, scale formation on the skin for 14 d. The mean marks were shown according to their score of brutality triggered by formulation applications noticed in table 3.

In vivo antifungal activity
Experimental procedure (in vivo), which includes the handling, was permitted in the proposal of the Committee of Control and Supervision of Experiments on Animals, No. 33/2019 approved by the (CPCSEA). The study aimed to investigate any symptoms that occurred due to the induction of fungal infection (Candida albicans) and improve the optimized formulation's efficacy. The hair on the dorsal side was shaved with the help of an electric clipper, and Veet cream was applied in this area for 5 min to remove the remaining hair and wiped that area of the skin with wet cotton and cleaned with alcohol, 4 h before starting the study. Infected 15 healthy female Wistar albino rats (200-250 g) were classified into five collections in every group (n=5) as is illustrated below: 1 st group: Negative control, 2 nd group: Placebo polymeric in situ film (not infected by Candida albicans suspension), and 3 rd group: Optimized VCZ in situ polymeric film.
The inoculation was performed under general anaesthesia. Around 3 cm 2 skins back to the examined rat were cleanly removed using an electric clipper, and they were marked respectively. Candida albicans suspension was adjusted to give (10 7 CFU/ml) was gently implied to rub the cleaned and shaved skin using a sterile, cottontipped swap till they were cleansed. They were not having any visible fluid applied to their shaved skin by gentle impression with a sterile, cotton-tipped swap until no more visible fluid was observed.
After the application of each formulation, the animals were kept individually in separate cages to prevent licking of the skin. Rats were provided with regular food and water and observed for any infection. The application of the drug started after the fungal growth was confirmed (5-d). Treatment was administered for 14 d for infected rats.
Additionally, clinical and microbiological characteristics were examined. These included clinical symptoms such as itching, redness, dryness, papules, and scaling on the skin, as well as the microbial findings include microscopic findings of papules, papulovesicular, scales, crusts [36,37]. The mean marks were shown according to their score of brutality triggered by formulation applications noticed in table 3. None erythema 0 2 Minor erythema (scarcely traceable bright pink) 1 3 Reasonable erythema (dark pink) 2 4 Modest to severe erythema (bright red) 3 5 Severe erythema (ultimate redness) 4 Histopathological examination was done for the skin tissues and organs of G1, G2, and G3 rats. In the last stage, the rats were immediately euthanized and sacrificed by the cervical dislocation method on the 15 th day. The treated part of the skin was collected from all the treated groups, and collected samples were sent to histopathological centre Clarette Biotech Private Limited, Mysuru, India.

Stability studies
The stability analysis is one of the essential planning variables to ensure protection and product adequacy. Meanwhile, it represents a significant advance on forms of improvement. It can show that the essence of the item influenced by various ecological elements changes over time to demonstrate the manufacturing industry potential. Drug degradation or decomposition occurs between the capacity conditions resulting from experimental drug or product instability changes. This could quickly decrease the centralized dosage form of the medicine, so the stability analysis was evaluated. The stability analysis aimed to estimate a product shelf-life by accelerating the degradation rate; in most cases, the temperature and relative humidity (RH) conditions were increased [38,39].
An optimized formulation was kept away from the light for six months. And checked every two months; various measures including viscosity, the volume of solution delivered for every actuation, pH, the angle of spray, spray pattern, and in vitro experiments on drug release were subject to the formulation. The study procedure used was identical to that mentioned previous section. It was necessary to separate the samples for the stability analysis into three different groups and store them in a refrigerator (4±2˚C), room temperature (25±2˚C/60±5 % RH), and humidity control chamber (40±2˚C/75±5% RH).

Design of experiment (DoE) for VCZ in situ film formulations
A 3 2 -level factorial, for optimization of the formulation, complete factor design was employed. The rate of the independent variables was based on the preliminary results of the batch. For the ERS, the low, medium, and high rates were 20%, 22%, and 24%, respectively. Plasticiser was used at low (2%), medium (3%), and high (4%) rates and VCZ was a constant value of 1 %. Respectively. Variable effect independently on Y1 viscosity (cps) and DT (s) Y2. Thus, to achieve the formulation with desired Viscosity and DT. Two independent variables were tested in the 3 2 -complete factorial design, each at three levels, and experimental trials were performed for all nine possible combinations [40]. The design layout of the 3 2 -full factorial design is shown in table 4. Two independent variables were selected as below: X2 = concentration % w/v of Sorbitol. Based on the experimental design, the factor combinations resulted in nine different polymeric film formulations; as per the suggestion, different polymeric film formulation batches were prepared and then evaluated for both respondents. The responses observed were fit to 9 runs, and it has been noted that the best fit model was a quadratic model for the two dependent variables. The model's significance and the model terms generated were analyzed by analysis of variance (ANOVA).

Drying time
The polymeric in situ film-forming drying time was determined to ascertain how quickly the film forms once the solution is sprayed. In situations of room temperature, the polymeric solution has been sprayed on the glass slide surface and then allowed it dry at ambient temperature [38]. The drying time was also directly observed after applying the polymeric film-forming solution on the skin (hand-arm), as illustrated in fig. {1 (ii)}. A glass plate was put against the film formed without being pressed to determine whether the film had dried. This technique represents realistic conditions because the skin has pores and body heat, causing the drying time in the film time drying test to differ from glass plates; additionally, a polymer film solution was applied to the skin of rats, as shown in fig. {1

Viscosity
Moderate viscosity is an acceptable flow characteristic of in situ filmforming solutions. High or low viscosity results in non-uniform film production; viscosity is one of the most significant rheological parameters for polymeric in situ film [40]. It affects the use of film and in vivo performance [38]. Viscosity is a significant factor in the time the drug resides in the skin. The viscosity of the polymeric film solution was found at 0.12, 0.12, 0.14, 0.13, 0.13, 0.14, 0.19, 0.19, and 0.20, respectively. Optimized formulation F5 displayed sufficient viscosity, which revealed a good film property of in situ film for topical application, and they were not highly viscous and easily flowable film. The viscosity profile of formulation F1 to F9 is shown in   fig. 3, which indicated a significant increase in the viscosity (R1). With an increase in the concentrations of components A and B.

Fig. 3: (a) Response surface plots of R1 Viscosity (cps) of VCZ based in situ film Formulation (b) Linear difference between occurrence and estimated viscosity value (c) Overlay plot viscosity on VCZ dependent in situ film formulation
Characterization of optimized polymeric in situ film formulation (F5)

FTIR spectral study
FT-IR spectroscopy was employed to obtain conformational information about the film-forming. It was used to investigate the interactions between polymer ERS, drug VCZ, and their physical mixture; the OH range VCZ of FTIR generally demonstrates absorption peaks around 3200, 3046, and 1090 cm-1, which are attributed to the stretching vibrations of OH, CH, and CO, respectively. The VCZ powder exhibited absorption peaks at 3200 cm-1, which corresponded to the stretching vibrations of OH. Bands at 3100-2850, 1670-1390, and 1360-1250 cm-1 were assigned to the alkane CH, C = C aromatic, and aryl C-N stretches. In a physical mixture of VCZ and ERS, the spectrum of the physical mixture of VCZ and ERS was identical. There is no presence or disappearance of VCZ peaks of the same characteristics as those listed in table 7 and verified by the lack of chemical interactions between VCZ and ERS. From the results, the compatibility of VCZ and the selected polymer were concluded in fig. 4.   In a physical mixture, the characteristic endothermic peaks of both drug and polymer were observed original authentic locations. VCZ had almost no interaction with the polymer. T°-melt begins, Tm-melting point, Tc-melting completion, DSC data collected at 10 °C/min.

Scanning electron microscopy
Morphological investigation of VCZ in situ film-forming optimized formulation (F-5) and Placebo in situ film-forming formulation (film dried) were carried out with the help of scanning electron microscopy (SEM). SEM images were illustrated in fig. 6 and showed that prepared in situ films of optimized formulation were spherical with a smooth surface. Particulate aggregation was not observed, and results were in good agreement with particle size analysis results. No residual, intact crystals of VCZ were detected on the in situ film surface, establishing that the in situ film matrix was entirely formed by VCZ and ERS 100.

Fig. 6: Scanned images of the blank polymeric film (A) and VCZ loaded polymeric film (B) displaying the surface morphology X-ray diffractometry
The crystalline character of pure VCZ, ERS, physical mixture, and in situ film dried was evaluated using X-ray diffraction, as illustrated in fig. 7

Rheological study
The purpose of flow testing is to determine whether a compound is thixotropic or not. If a mixture has these flow characteristics, it will pass through the sprayer nozzle repeatedly. The following property makes the film-forming solution being thinner as it passes through the nozzle (stressed) and returns to its initial viscosity when sprayed (stress is lost). For any formulation, it is important to test their rheological behavior for topical applications of drug delivery. For its effectiveness, the delivery of molecules in or through the skin is essential. The acceptable film-forming solution flow characteristic is moderate viscosity, as high or low viscosity contributes to non-uniform film production [39]. The relationship among the shear stress and shear rate can be explained by the Ostwald-de Waele model or power-law equation as mentioned below: Where σ Indicates the shear stress (Pa), K indicates the consistency index (Pa s n ), γ Indicates the shear rate (s −1 ), and n indicates the flow behavior index.  fig. {9 (i)} and Placebo formulation in fig. {9 (ii)}). For the Newtonian flow-type, # n is always 1

In vitro release review of cellophane membrane
Studies of the drug release were performed in vitro using a cellophane membrane in a Franz diffusion cell. It includes a donor cell and a receiver compartment. The receptor compartment was filled in as a diffusion medium with 20 ml of phosphate buffer pH 7.5 solutions. In the receptor compartment, the prepared polymer solution was a spray, magnetic beads were used to stir the liquid constantly at 50 rpm, and 37±1 °C held its temperature. 2 ml receptor fluid sample was withdrawn at fixed intervals. The same amount of 2 ml phosphate buffer solution was replaced with the samples measuring their absorbance at 256 nm. The total volume of drug release transported in a formulation containing 22% of ERS and 1% of VCZ was 99.33±0.07 percent measured from the slope of the linear portion of the curve in table 10 and fig. 10. Mean value (±SD) n=3

Ex-vivo drug diffusion studies
In situ topical polymeric film formulation F-5 was chosen to study Ex-vivo permeation because it provided the best release among all other formulations in an in vitro drug release study. The di ffusion studies from the optimized formulation F5 were performed out over 7 h. Because of its ex vivo similarity to human skin [41,42]. Rat abdominal skin was used as an animal model skin. The sample obtained was analyzed by a UV spectrophotometer at 258 nm. Table  11 and fig. 11 show the release pattern of optimized formulation F5. The formulation F-5 exhibited a significantly high penetration of VCZ in situ polymeric film. The total amount of VCZ in situ polymeric film transported was found to have a cumulative drug release % of 97.02%.

Kinetics data analysis
Ex vivo kinetics studies of VCZ in situ polymeric film transport data of optimized formulation were added analyzed. The model fit-up was carried out using internally built software from the excel sheet program (table 12). The cumulative percentage of VCZ permeated from in situ polymeric film via rat skin was obtained by plotting against time. It was confirmed to kinetics model (Zero-order; first order, Higuchi and Korsmeyer-Pappas) [43]. The best fit model was chosen based on the relatively high correlation coefficient and the correlation value, as described in table 13. A higher R2 value lowers fisher's rates were observed (0.9892 and 0.308, respectively). The result showed that the drug release of optimized VCZ in situ polymeric film follows the First order model, indicating the mechanism of drug release. It was more than 0.9892 multiple linear regression coefficient data for correlation of data noticed in fig. 12.

Skin irritation studies
A skin irritation study on rats' skin was performed to confirm the safety of optimized VCZ in situ polymeric film spray formulation. The irritation potential of the topical formulation was evaluated. According to the findings of this investigation, optimized VCZ in situ polymeric film spray formulation F5 did not cause substantial irritation (erythema, edema, itching, redness, dryness, papule, and scale) or inflammation on or around the site of application on rat skin for up to 14 d. In short, there were no apparent signs of skin irritation (erythema and edema) observed on visual observation of skin specimens treated with VCZ loaded in situ polymeric film formulation indicating the absence of any skin irritation. Fig. 14 demonstrates the findings.

Histopathological studies
Skin specimens were investigated to evaluate cutaneous condition potential of optimized VCZ in situ polymeric film, placebo in situ polymeric film, and untreated rat skin (negative control). Histopathological examination for treated rat skin by VCZ in situ polymeric film was observed normalized epidermis and provisional matrix, well-defined collagen, and Elastin layer or well-defined dermis with capillaries. There was a prominent boundary between the provisional matrix and mature dermis; microvasculature was appeared and shown in (fig. 16{A1}).
Ratskin treated by placebo in situ polymeric film was observed; in the epidermal layer, the photograph showed a decrease in the size of the sebaceous gland, with a flattening thick, horny epidermal layer, and hair follicles with a narrow lumen. Dermis fibers and hypodermis were poorly identified with collagen, elastin fibers. Collagen fibers lost their details and were combined with a homogenous, hyalinization appearance. Microvasculature is found in a provisional matrix, as depicted in fig. 16{A2}.
The skin was seen with photomicrographs of the negative control. The picture showed hyperkeratosis in the epidermal layer with a flattening thick horny layer, a decline in sebaceous gland size, and hair follicles with a narrow lumen. Collagen, elastin fibers of the dermis and hypodermis of the showed poor-defined. The collagen fibers lost their details and appeared fused with a homogeneous, hyalinization appearance was found. Microvasculature found in the provisional matrix indicated by an arrow, as shown in fig. 16{A3}.
Histopathological studies have shown that VCZ, a topical in situ polymer film spray, is not irritating or affecting the skin. No liaisons were observed; neither was any significant change in the skin or any break in the epithelium's length. No ulceration was seen.
Moreover, the results of significant organ histopathology examinations of liver and kidney fig. 16 {B and C} revealed no damaging effects from repeated skin exposure to any of the groups. Insignificant alterations were detected as a result of the findings. This could be related to the reasons that only a tiny amount of the drug is absorbed into the blood circulation upon topical treatment of VCZ from an in situ polymeric spray solution or a placebo formulation. Hence, VCZ topical in situ polymeric film sprays may be considered a safe option for the skin.

Stability studies
The results of the stability study of the optimized VCZ in situ polymeric film formulation (F-5) after one month, two months, and six months at different storage conditions (in the refrigerator (4±2 ᵒC), room temperature (25±2 ᵒC/60±5% RH) and humidity control chamber (45±2 ᵒC/75±5% RH)) are shown in

ACKNOWLEDGEMENT
The researchers profoundly put their words of gratefulness and thankfulness for the JSS Academy of Higher Education and Research, Mysuru, 570015, Karnataka, India, for their significant facilities and constant support for carrying out the current study.

ETHICAL APPROVAL
All experimental regulations were directed and applied based on the study's ethical procedures approved by the Institutional Animal Ethical Committee recognized, JSS College of Pharmacy, JSS Academy of Higher Education and Research, Mysuru, India.

AUTHORS CONTRIBUTIONS
All the authors have contributed equally.