Application of DoE in polymers screening and optimization of in situ topical ϑilm-forming solution for spray formulation

DoE is a structured and organised method to determine the relationship between the effect of change in the concentration of the independent variables and its impact on the formulation, through establishing amathematicalmodel. Since the acceptance of the QbD approach by the regulatory authorities across the world, DoE has been widely implemented in the areas of screening and optimisation of the formulations by the pharmaceutical industries. The topical delivery of API still posses’ limitations such as insuf icient contact time, odd hours of application time, sticking to fabrics, formulationwashing off, etc. To address these limitations, the researcher planned to develop an in situpolymeric sprays that will form a transparent and lexible ilm, &will not interfere with the applicant’s routine. Polymers such as HPMC, Eudragit RS100, PVP K30, PVP K90, Carbopol, Propylene glycol, Soluplus, and pullulan whereas the plasticisers selected were sorbitol. Voriconazole, a second-generation triazole, was used as amodel drug. The article is a technological demonstration, in which the screening of polymers as well as the optimised concentration of the polymeric will be selected through 3 factorial design. The aim of the present article is also to establish the relationship between the software response and experimental values. The experimentswere designedusing 3 factorial design which resulted in 9 trial runs. Each run was evaluated for drying time, viscosity, and stickiness. The resultant response surface Later the optimisation, to yield an optimised polymeric solution that can deliver a desired in situ ilms. Based on ANOVA comparison of variability due to treatment, the signi icance of the regression model was evaluated. Other procedures such as DSC, FRIR, Stickiness, pH, diffusion studies were also performed on the selected formulation.


DoE,
QbD, 32 Full Factorial Design, ANOVA, In Situ Film, Drying time, viscosity A DoE is a structured and organised method to determine the relationship between the effect of change in the concentration of the independent variables and its impact on the formulation, through establishing a mathematical model. Since the acceptance of the QbD approach by the regulatory authorities across the world, DoE has been widely implemented in the areas of screening and optimisation of the formulations by the pharmaceutical industries. The topical delivery of API still posses' limitations such as insuf icient contact time, odd hours of application time, sticking to fabrics, formulation washing off, etc. To address these limitations, the researcher planned to develop an in situ polymeric sprays that will form a transparent and lexible ilm, & will not interfere with the applicant's routine. Polymers such as HPMC, Eudragit RS100, PVP K30, PVP K90, Carbopol, Propylene glycol, Soluplus, and pullulan whereas the plasticisers selected were sorbitol. Voriconazole, a second-generation triazole, was used as a model drug. The article is a technological demonstration, in which the screening of polymers as well as the optimised concentration of the polymeric will be selected through 3 2 factorial design. The aim of the present article is also to establish the relationship between the software response and experimental values. The experiments were designed using 3 2 factorial design which resulted in 9 trial runs. Each run was evaluated for drying time, viscosity, and stickiness. The resultant response surface Later the optimisation, to yield an optimised polymeric solution that can deliver a desired in situ ilms. Based on ANOVA comparison of variability due to treatment, the signi icance of the regression model was evaluated. Other procedures such as DSC, FRIR, Stickiness, pH, diffusion studies were also performed on the selected formulation.

INTRODUCTION
Polymeric ilms form an intact layer with the skin surface and hold the APIs for a longer duration of time by resisting any modi ication even by rubbing and washing (Mcauley et al., 2015). They are easy to apply and are devoid of any complications systemically observed in dosage and topical semi-solid formulations. Polymeric ilms are designed so that they produce desired effects, easy to apply with improved patient acceptance (Frederiksen et al., 2016).
In situ polymeric ilm solution is a novel approach that may produce the unconventional dosage formu-lae applied on the skin like ointments, creams, gels, spray, and patches. In situ ilm polymeric solution spray is applied in the form of liquid which forms a transparent ilm in situ after quick evaporation of the solvent. Polymeric solutions forming ilms are widely used in surgery, wound care and protection of skin. In surgery, these are used for sealing incisions without using sutures and act as tissue glue or as disinfectants during pre-operative preparation. Topical polymeric ilm solutions are also used for treating minor cuts, abrasions or for protecting the ostomy wound from surrounding body luids, and they may or may not contain antimicrobial substances (Kathe and Kathpalia, 2017).
Polymers are the base for ilm-forming solutions, and different types of polymers are used for the preparation of these solutions. Polymers can be used alone or in conjunction with other polymers to achieve the desired result. The polymers must have the ability to form a clear transparent ilm at skin temperature upon application. Film formation using polymeric solutions is a comparatively simple process as the polymer is already in the liquid state. Polymer chains start interpenetrating at a specifying concentration and viscosity when the droplets start vaporising from the substrate surface 9 . The polymeric network formed on the substrate controls the release of the drug constituent by serving as an external reservoir. It reduces the excessive release of drug substance to the skin reservoir (Tran and Tran, 2019).
The research aims to design and characterise antifungal in situ ilm-forming polymeric solution spray containing Voriconazole for topical drug delivery. The work focuses on a wider variety of polymers, on selecting suitable polymers and on characterising the properties of the resulting formulations so the production of this novel in situ polymer solution spray as a dosing form can be focused on a broader technical basis.

Preparation of Voriconazole in situ ilm
Steps associated with the formulation of polymer in situ ilms, as shown in Figure and Composition of polymeric topical delivery formulations, as shown in Figure 1.

Determination of solubility
Voriconazole saturation solubility was measured in the pH 1.2 HCl, Phosphate pH buffer 6.8, Phosphate pH buffer 6.8, ethanol and distilled water. Each media was prepared, and excess samples were applied to 25 ml of each medium put in the conical 50 ml lask and held for 24 h for shaking on mechanical shake. After 24 h of shaking, 1 ml of aliquot was removed from each sample and iltered through No. 0.45 micron, whattman ilter paper. Absorption has been calculated in the 200-400 nm range of visible UV spectrophotometer, and solubility measurements have been carried out (Parthibarajan et al., 2012).

Differential Scanning Colorimetry (DSC)
The pure drug (Voriconazole) and physical mixture with polymer (Eudragite RS100) thermal characteristics were done by differential calorimeter scanning (Shimadzu, japan, DSC-60) mention in the table and igure. Sample with about 5 mg was placed in aluminium pans for each sample on it, and DSC analysis was performed at a low amount of 20 ml min −1 . Dynamic scans were rendered within the temperature range of 10 to 300 • C in the nitrogen atmosphere at 10 • C / min. Indium has been used as a standard reference for temperature adjustment (Schroeder et al., 2007).

FTIR Spectral Analysis
The pure drug (Voriconazole), polymer (Eudragite RS100) and their physical mixture of the Fouriertransform (FTIR) spectra were recorded using a visible UV spectrophotometer (FTIR-8400S, Shimadzu, Kyoto, Japan) and mentioned in the table and igure. Disks of each sample (5 mg) were individually mixed with 200 mg of potassium bromide (spectrographic rank) and compressed into the disks using a hydraulic press (4000 cm −1 to 400 cm −1 ) before scanning (Misra et al., 1996).

Film Forming Capacity of Various Polymers
The polymeric placebo ilms were prepared using the technique of solvent casting. The polymeric placebo ilms were prepared using various solvents                   such as ethanol, and hydroalcoholic solution. The purpose of the experiment was to research the impact of solvent on the ilm-forming capacity of the polymers without plasticisers. Solvent effects and ilm quality were studied in terms of transparency, ilm stickiness, and drying time. The different types of polymers like as Eudragit RS100, Polyvinyl pyrrolidine K30, Polyvinyl pyrrolidine K 90, Hydroxypropyl Methylcellulose, Carbopol, Pullulan, Soluplus, Propylene glycol, were investigated for their ilm-forming ability given in Table 1.

DoE for optimising formulation
For the optimisation of the formulated polymeric ilm, a full 3 2 design factory was used. The concentration of polymer (X1 Eudragit RS100) and plasti-ciser (X2 sorbitol) was chosen as independent variables according to the literature review, and it is shown in Table 2, Viscosity (Y1) and dry time (DT %) (Y2) were chosen as dependent variables, and API was a constant value. A statistical model is concerned with compositional response (degradation) Equation (1).
It is proved that Y is the interaction response, whereas b0 is the arithmetic uncaring overreaction of the nine trails. The above interaction in the equation Y is the measurable result of preparation ingredient or different autonomous changes X and Y; b0 is the average arithmetic interaction; b1, b2 and b3 are the correlated coef icients for the elements  (Osmani et al., 2016).

Characterisation of Voriconazole polymeric ilm formulations
Typical control tests in topical ilm polymeric solutions are designed to optimise and improve the delivery system and formula pro iciency.

Drying time (DT)
The solution was sprayed on the skin surface, allowing drying and recording the time. In a normal environment, solution drying can be done, and the ilm is formed.

Morphological characterisation of the ilms
The outer surface of the dried ilms was evaluated for its stickiness/ smoothness by pressing and absorptive rubbing cotton wool under low manual pressure on the dry ilm, after pressing and rubbing of cotton wool, if the ibres from the cotton wool stick to the outer surface of the ilm then it was termed as sticky. If no ibres were found, the outer surface was said to be smooth (Saudagar, 2014).

Determination of Transparency
The transparency of the dried ilms forming was established by visual inspection and graded according to Table 3. The ilms were also inspected for any bubble entrapment a deformation.

pH
Digital pH meters were used to determine the pH of the polymeric spray solution. The phosphate buffer pH meter of 7.4 has been calibrated. The pH electrode was dipped to reach a pH of 20 ml of polymeric solution spray in a little beaker of glass. The pH was determined three times in every formula, and average values were estimated (Padula et al., 2019).

Uniform Thickness of Films
The ilm-forming thickness was measured using a 0.01 mm precision handheld micrometre (Dial Gauge thickness 7301, Mitutoyo, Kanagawa, Japan). The thickness of the ilm samples was measured at 4 to 6 random points, and the mean value was used (Gohel and Nagori, 2009).

Viscosity
The solutions were measured with Brook ield viscometer (Brook ield engineering laboratories, Inc., USA.) at a viscosity of 25 ± 1 º C. The spindle ULA S00 was rotated at 10 rpm, and 20 ml of the sample was taken in ULA cylinder. The torque readings were still higher than 10%. Three measurements were taken, and the average was determined.
AL-the quantity of the sample given at each distribution, Wt-after distribution of formula weight, Woinitial formulation weight before distribution, and Dn-density.

Quantity of solution given for each actuation
Amount of solution supplied with every operation. Using the volume of the polymeric ilm solution provided at each actuation was calculated (Pawar et al., 2017).
Where, A L -The quantity of solution given at every actuation, Wt-Weight after-action formulation, Woinitial formulation weight before a performance, and Dn-formula's density.

Density
The dried and emptied Pycnometer was weighed and illed with the sample. The air bubble was permitted to rise to the top before inserting the stopper. The right value of the density was calculated by dividing the inal liquid weight in Pycnometer (Ranade et al., 2017).

The angle of the spray
The spray of solutions was stimulated latly onto a white paper attached at 10 cm from the nozzle. A circle is formed on the paper, the radius of which is noted down. Radius from different directions was registered three times. The angle of spray (θ) was calculated by Eq =tan −1 (1/ r).
Where 1-the distance between the paper and the nozzle, r-Median circular radius.

The pattern of the spray
For the study, the method used to impinge spraying on a piece of paper. 10 mg Methylene blue was dissolved in the polymeric ilm solution to facilitate visualization (Mustapha et al., 2016).

Folding Endurance
Folding stamina was speci ic by repeated ilm folding at the same location until the ilm breaks. That indicates the ilm's brittleness. It is measured as the amount of folding endurance as the number of times when the ilm is plied without breaking. In the paper test, the folding endurance is the logarithm (to the base of ten) of the number of double folding parts necessary for breaking the test part under standardised conditions (for Testing and Materials, 2002). F = log 10 d Where F is the folding endurance; d the number of double folds.

Tensile strength
The tensile strength was a ilm strength measurement and is calculated by dividing the maximum force required to break the ilms by the ilm cross-sectional area. To determine the peak load and tensile strength of the ilm, a universal test machine (International Equipment's, Mumbai) was used (Anter et al., 2018). The result is expressed in megapascals (MPa) and registered to a signi icant character.

Elongation at break %
The per cent elongation was determined by dividing the elongation by the initial gauge length at the moment of the rupture and multiplying by 100. When using gauge marks or extensometers to de ine a particular section of the test. Tensile testing was used to determine the ilm peak charge, elongation at peak load. The tested ilm was determined as follows (Kaur et al., 2013).
L0 × 100 Where L is the ilm's inal length and where L 0 its original length.

Water Vapor Permeability
The permeability of the ilm samples to water vapour was calculated gravimetrically by the process. In WVP tests, ilms were chosen based on a lack of physical defects like cracks, bubbles and pinholes. Horizontally placed rectangular ilm samples (5 mm / 5 mm) on the WVP weighed in 10 ml beakers and illed with distilled water up to 1 cm below the picture. The beakers were set at 25 o C and 50 per percentage RH in the regulated humidity chamber with 1 m / min air current circulation. For a period of 8 and 24 hours, the cup was weighed every hour. The transmission rate of the water vapour (WVTR) was estimated from the slopes of the cup's steady-state portion of weight loss versus the time curve (Abdellatif and Tawfeek, 2016).

W V P R = W
A * t (g/cm 2 * 24 h) Where WVTR is the calculated rate of water vapour transmission (g / m 2 s) through with a ilm, A was ilm surface area, and t was the time (h).

Moisture content
The polymeric ilms (2 cm 2 ) were held for three days in a desiccator that contained activated silica and weighed (W i ) at room temp. The ilms were weighed periodically until we obtained a constant weight (W f ). The calculation of the (per cent) moisture content was based on the following equation (Maghraby et al., 2008).
Where W i is the ilm's initial mass and W f is the ilm's inal weight.

In-vitro release review of cellophane membrane
The modi ied Franz diffusion cell was used as a cellophane membrane for the permeation test. The cellophane membrane was immersed overnight in distilled water. Then it was soaked 24h before usage in pH 7.4 stock solution. The cellophane membrane was between the donor cell and the recipient cell. The spray of a polymeric solution was moved to the donor cell. The entire membrane surface was in contact with a receptor compartment containing 20 ml of pH 7.4 buffer of phosphate 19 . The cell was agitated at 50 rpm on a magnetic stirrer and kept at 37±1 • C. Every time 1 ml was withdrawn at intervals of 15, 30, 60, 120, 180, 240, 300, 360, and 420 min and replaced with the same fresh buffer of phosphate pH 7.4.

Stability Studies
Optimised formulation (batch F5) was kept away from the light for six months at 30 ± 3 2C. And checked every tow month, Various measures including viscosity, the volume of solution delivered for every actuation, pH, angle of spray, the pattern of spray and in vitro experiments of drug release were subjected to the formulation. The study procedure used was identical to that mentioned previous section (M et al., 2020) .

Determination of solubility
The results of Voriconazole's qualitative solubility in various solvents are shown in Table 4 and Figure 2 below.       Figure 3 show the respective voriconazole DSC thermograms and their physical mixture (Voriconazole with Eudragite RS 100). Voriconazole thermogram exhibited a broad endothermic peak at 138.07 • C related to the evolution of the sample's moisture. The prominent melting peak at 182.91 • C manifested the natural crystalline state of polymer (Eudragite RS 100). The characteristic endothermic peaks of both drug and polymer were observed at their authentic locations in the case of a physical mixture, and there was almost no interaction between Voriconazole and polymer.

FTIR Spectral Analysis
FT-IR spectra of Voriconazole, polymer (Eudragite RS 100) and its physical mixture (Voriconazole + Eudragite RS 100), The range of OH Voriconazole FTIR generally exhibited stretching at 3200.09-3046.04 cm −1 , C-N extending at 1510.28-1451.28 cm −1 , and C-F at 1587.44-1451.28 cm −1 , shown in Figure 4, respectively. The Eudragite RS-100 FTIR spectrum showed a peak of 2953.9 cm-1 due to the presence of O -H (carboxylic acid), 1450.7 cm-1 due to the -CH2 bend, and 1731.2 cm-1 due to the presence of C = O (ester). The spectrum of the physical mixture of the drug (Voriconazole) and polymer (Eudragite RS 100) was identical, in a physical mixture of Voriconazole and polymer, no presence or disappearance of voriconazole peaks of the same characteristics as the ones listed in Table 6 and veri ied by the lack of chemical interactions between Voriconazole and polymer.

Characterisation of placebo ilm-forming polymers
Characterisation of placebo polymeric ilm-forming solution with absolute ethanol it's given in Table 7 and Figure 5.
Characterisation of placebo polymeric ilm of a ilmforming solution with a hydroalcoholic solution it's given in Table 8, Figure 6.

DoE for optimising formulation
A 3 2 -level factorial, for optimisation of the formulation, complete factor design was used. The rates of the autonomous variables were based on the preliminary results of the batch. The low, medium and high rates were 20%, 22% and 24% respectively for Eudragite RS100. Plasticiser was used at low (2%), medium (3%) and high (4%) rates, respectively. Variable effect independently on Y1 (cps) viscosity and time of drying Y2 (seconds). The equation ANOVA indicated the model F values 760,20, P-value < 0,0001; showing the model to be signi icant. ANOVA for drying time and viscosity conclusion mentioned in Tables 9, 10 and 11. The reduced model was confounding, but the most important parameters in luencing the response were screened (Eq. 1).
2644 -254.42 X 1 + 15 X 2 + 6.12 X 1 -2 X 2 The correlation coef icient was respectively 0.995 and 0.993 in the complete model and reduced form. Increased Eudragit RS 100 concentration increased drying time requires optimised formulation (Y1) development mention in Figures 7, 8 and 9. The polynomial equation above shows a strong match of response variables at various levels.
For both the full model and reduction model the correlation coef icient value was 0.859 and 0.810 respectively. Y2 (Viscosity) was affected as a major factor by the increased concentration of polymers (Eq. 2). 2.44 -0.23 X 1 + 0.00 + X 2 + 0.01 + X 1 -2 X 2 note in Figures 10, 11 and 12.

Characterisation parameters related to polymeric ilm formulations
Forms of evaluation ilms for all formulations such as drying time, transparency, stickiness, lexibility and structural characteristics. After analysing all the formulations, in terms of the ilm-shaped and unregulated from the negatives and defects present in some of the formulas are shown in Table 12 and in Figures 13 and 14 the best suitable. pH pH measurements were made using a pH meter. For measuring pH, 20ml Spray for the solution was used. Results are shown in Table 13 and Figure 15.

Thickness
The thickness of all formulations was between 0.01±0.03 to 0.08±0.02 their mention in Table 14.

Water Vapor Permeability (WVP)
The results of studies on the transmission rate of water vapour (WVTR) are shown in Table 15. And then in Figure 16. Also, plasticiser addition was found to affect the permeability of polymer ilm as the experimental humidity conditions affected the WVTR, 25 per cent humidity conditions, and 50 per cent throughout the study were employed. All polymeric ilms showed low WVTR indicative of its hydrophobic character. A decrease in WVP is considered advantageous for application to skin targets.

Moisture content
Studies of the moisture content gave an insight concerning the stability of the ilm. The moisture content (percentage) of polymer ilms was low (1± 0.113 per cent, 1± 0.113 per cent, 1± 0.113 per cent, 1.5± 113 per cent, 1.5± 0.113 per cent, 2. ± 113 per cent, 2±11 per cent, 2.05± 12 per cent, 2.5± 10 per cent) respectively, which may help preserve stability and prevent dryness and fragility during long-term storage, especially under dry costorage, noted in Table 16 and Figure 17.

Viscosity
Formulations viscosity containing Eudragite RS100 as a polymer and glycerol as a plasticiser, formulations viscosity results were noted in Table 17 and Figure 18. and suf icient spray capability is demonstrated.

Quantity of solution given for each actuation
According to the equation given in the evaluation, the result will be as follows. For each actuation, the quantity of solution was 0.289±0.021 ml.

Spray angle
Spray angle was evaluated from local container spray and equation calculated by =tan −1 (1/ r). Where 1-the distance between the paper and the nozzle, r-Median circular radius. Average radius circle = 2.2 cm and distance of paper from nozzle was15cm so, tan −1 = 15 2.2 = 6.8 cm tan −1 = 81.63 spray angle was 81.63 0 ±0.07

Spray pattern
It was observed that the formulation of the spray pattern was transparent, clear, spherical and uniform. After application of a clear transparent ilm, the drying time was 43 sec. The optimised formulation showed good sprayed properties. Figure 19 depicts an illustration of the ilms formed after drying (with the inclusion of methylene blue to improve visibility).

Folding Endurance
The values of folding endurance of formulation developed by the polymeric ilm were found in 80 folds which are considered adequate, elastic and reveal good ilm quality.

Tensile strength
Tensile strength was determined to de ine polymer ilm for its abrasion resistance and durability, the formula determined the tensile strength.  Table 18.

Elongation at break %
Due to their abrasion resistance and durability, elongation at break was determined to characterise polymer ilm, noticed in Figure 20.

In vitro release study using cellophane membrane
Studies of the release of the drug were performed in vitro using a cellophane membrane in Franz diffusion cell. It includes a donor cell and a receiver compartment. The receptor compartment was illed in as a diffusion medium with 20ml of phosphate buffer pH 7.5 solution. In the receptor compartment, the prepared polymer solution was a spray, Magnetic beads used to stir the liquid constantly with 50 rpm, and 37±1 • C held its temperature. 2ml receptor luid sample was withdrawn at ixed intervals, and the same amount of 2ml 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 Eudragite RS 100 and 1% of Voriconazole was 101±0.07 per cent by measured from the slope of the linear portion of the curve found in Table 19 and Figures 21 and 22.

Stability Studies
Optimised formulation F5 batch short-term stability test was conducted at 30±2 • C at room temperature for six months. Table 20 Shows that pH, viscosity, performance volume, spray angle, spray pattern, folding endurance, thickness, tensile strength, break per cent elongation, density, and in vitro release study the physical appearance of optimised formulation batch F5 remained unchanged through the study reference.

Discussion
The polymeric ilm-forming solution was prepared by dissolving selected polymers in different solvents such as ethanol, hydroalcoholic solution (1:1) and distilled water. The ilms formed upon drying were subjected to various preliminary evaluation like drying time, transparency and stickiness. The ilms prepared using absolute ethanol solvent with plasticiser showed satisfactory results when considering the drying time and visual observation. The polymeric ilm solution prepared using sorbitol as a plasticiser, which was selected based on the exhaustive literature search performed. It can be concluded that, the polymeric solution that was prepared using as a solvent was considered to be un it for the incorporation of the API because the ilm took a long time for drying and one of the formulations did not form a ilm.
The polymeric ilm formed using ethanol as solvent F5 showed the best drying time and was considered as optimiser formulation.
Formed ilms were prepared and evaluated. The drug used in these ilm formulations was Voriconazole. Out of 9 th formulations, 5 th formulation is best and gives aspired results from the evaluation con-ductedFigure 23 .

CONCLUSIONS
This study concludes that Voriconazole can form a topical in situ ilm-forming development by using a mixture of Eudragite RS100 and Sorbitol as plasticiser using the response surface method, and mentioned below results work. The impact of formulating variables on product properties can be easily predicted by using a 3 2 -level factorial experimental design and quadratic mathematical equations developed. Based on the speci ications of the product, such as Drying time (DT %), Transparency, Stickiness, Structural features of the ilm formed, Viscosity, Spray angle, Spray pattern, Mechanical properties of the polymeric ilm formed and in vitro using a cellophane membrane, the best batch of topical Voriconazole in situ ilm-forming would be 22% Eudragite RS100 and 4% Sorbitol. It can be concluded. mal delivery of terbina ine hydrochloride. Int J Pharm Sci Res, 5(9):537-54. Schroeder, I. Z., Franke, P., Schaefer, U. F., Lehr, C.-M. 2007. Development and characterization of ilm forming polymeric solutions for skin drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 65 (1)