DESIGN, FABRICATION, IN VITRO , AND EX-VIVO PERMEATION STUDY OF MICRO-EMULSIFIED HYDROGEL OF FLUCONAZOLE (MHG-FLCZ) USING A CENTRAL COMPOSITE DESIGN (CCD)

Objective: The current study's objective was to develop and characterize a micro-hydrogel-based fluconazole (FLCZ) gel. A micro-hydrogel (Mhg) was prepared using different concentrations of Carbopol 940 (CP) and NaCMC using the modified swelling hydrogel method. Methods: A Preformulation study was performed using FTIR to confirm the drug and polymers were compatible with each other based on the functional group determination. 3 2 optimization procedures were used to develop formulations based on the response surface methodology. The prepared formulations were evaluated for entrapment efficiency, spreadability, viscosity, and visual examination using binocular microscopy and in vitro drug release using Franz diffusion cells. Results: The optimized formulation F2 reported entrapment efficiency of 65.09±0.41%, and viscosity of 11100±1.21 cps. The in vitro release of drug for the prepared formulations was performed for 8 h. and the optimized formulation showed better-controlled drug release compared to other formulations. It was observed that the optimized batch, percentage of drug permeability through the skin at 8 h of ex-vivo study shows 84.67±0.67% and in vitro drug release study (93.22%) through Franz diffusion cell, which suggests that the drug (Optimum batch) can easily penetrate through the skin and showed the highest drug release in a stipulated time interval. Conclusion: The use of an optimized Mhg-FLCZ gel formulation as it has excellent homogeneity, a pH that is close to that of the skin, and suitable thixotropic characteristics relates to that much more convenience than the conventional dosage form. The in vitro and ex-vivo study data proved its suitability as a better alternative to conventional products in the effective treatment of skin infections.


INTRODUCTION
Targeting the drug delivery system to the topical route is much more prominent in delivering the dosage form to avoid first-pass metabolism, enzyme degradation by the gastrointestinal tract, and improved patient compliance [1].Topical formulations are smeared, rubbed, sprayed, or instilled directly onto the external body surface.Both local and systemic medication effects have been produced by using the topical route of administration to treat skin diseases [2,3].Fluconazole (FLCZ), an antifungal agent belonging to the azole group of drugs interacts with the enzymes responsible for ergosterol synthesis which alters the cell membrane fluidity.This micro-hydrogel improves drug solubility and facilitates drug absorption across the skin in comparison to the traditional topical preparation [4].The gel is emollient, promptly spreadable, quickly removed, non-staining, and readily compatible with several types of excipients.An essential feature of gel systems is the str uctural network formed by polymers.Gel formulations show variation with the variability of polymer type and concentration, which affect drug release [5].The term micro-emulsified hydrogel (Mhg) describes thermodynamically stable, fluid, translucent, or transparent homogenous dispersions composed of the oil phase, aqueous phase, surfactant, and co-surfactant in the appropriate ratios, a single optically isotropic in nature dispersion with a droplet diameter usually in the 10-100 micrometer range is generated [6].Micro-hydrogel (Mhg) is far superior to conventional formulations because it enhances drug solubility, is easier to manufacture, and is more effective at delivering drugs transdermally [7].
The current study's objective was to design and characterize a micro-emulsified hydrogel of fluconazole (Mhg-FLCZ) using a central composite design (CCD).In order to formulate a Mhg-FLCZ, carbopol 940 (CP) and sodium carboxymethyl cellulose (NaCMC) at different concentrations were used.3 2 optimization techniques were used to develop formulations based on the response surface methodology.The prepared formulations were investigated to determine the optimized formulation as entrapment efficiency, spreadability, viscosity, and visual examination using binocular microscopy, in vitro drug release, and ex-vivo permeation study using Franz diffusion cells.

Material
Fluconazole drug was gifted from Cipla, Sikkim, India.Carbopol 940 was purchased from Sigma Aldrich, America.Sodium carboxy methyl cellulose (NaCMC) and Glycerine were purchased from Sigma Chemical Corporation Pvt. Ltd.Other than that, all of the chemicals and reagents used in the studies followed pharmaceutical standards and were of analytical quality.

Fabrication of Mhg-FLCZ
All 9 formulations of FLCZ topical gel were prepared using different concentrations of Carbopol 940 (CP) and NaCMC using the modified swelling hydrogel method [8,9].The composition of different formulations of Mhg-FLCZ is presented in table 1. CP and NaCMC were soaked overnight at different concentrations.The organic phase was prepared using a drug (FLCZ) dissolved in 10 ml of methanol and glycerin.Organic phase transfer drop by drop through a micropipette to an aqueous phase containing CP and NaCMC makes oil in water (o/w) using a magnetic stirrer with the speed of 8000 rpm.Alcohol (methanol) was utilized to improve penetration while glycerin served as a moisturizing ingredient.Allow to hydrate and swell slowly addition of methyl paraben and propyl paraben sodium under stirring of 8000 rpm.To adjust the skin pH, triethanolamine was used and the content was overnight at room temperature to get a homogenous gel of Mhg-FLCZ.

Entrapment efficiency (%) of Mhg-FLCZ
The concentration of free drug in the dispersion medium was measured in order to assess the entrapment efficiency (%) (%) of different formulations of Mhg-FLCZ.10 μg/ml diluted solution of Mhg-FLCZ was prepared using phosphate buffer 7.4 and poured onto Whatman filter paper with a pore size range of 0.22 nm and collected the filtrate.At 260 nm, the absorbance of the solution was measured using a spectrophotometer (Shimadzu 1800).The equation entrapment efficiency (%) was used to calculate the entrapment efficiency of Mhg-FLCZ [12].
Entrapment efficiency (%) = Amount of drug used in the formulation − Amount of unentrapped drug Amount of the drug used in the formulation × 100

pH of Mhg-FLCZ
Mhg-FLCZ of each gel formulation was transferred to 10 ml of the beaker and measured by using the digital pH meter (Remi, India).pH of the topical gel formulation should be between 3-9 to treat the skin infections [13].

Spreadability of Mhg-FLCZ
Two slides (split into squares with 5 mm sides) were used to press a sample of 0.5 g of each formula between them.and when no further spreading was anticipated, left for around five minutes.The spread circle diameters were measured in centimeters and used as standards for spreadability [14].

Viscosity of Mhg-FLCZ
A Brookfield viscometer DVII model with a T-Bar spindle and helipath stand was used to determine gel viscosity.All Mhg-FLCZ gels were measured using Spindle T 95.T95, the T-bar spindle, was lowered perpendicularly to ensure that it wouldn't contact the bottom of the jar and measure the viscosity of Mhg-FLCZ formulations [15].

Measurement of viscosity of Mhg-FLCZ
Several factors impact this, such as sample size, sample pressure, and temperature.Viscosities were measured at several locations along the helipath by moving the T-bar spindle up and down [16].The torque value was consistently higher than 10%.The viscosity of gels was determined by taking an average of three readings in a minute [17,18].

FT-TR (Fourier transform infra-Red)
Fourier transform infrared spectroscopy (Perkin Elmer, America) was used to analyze the drug's interaction with the polymers of Mhg-FLCZ.FLCZ IR absorption peak was measured between 400 and 4000 cm -1 .utilizing the KBr disc approach [19].For the purpose of evaluating purity, the primary peak was reported [20].

Visual examination of Mhg-FLCZ
To determine the surface of the particles of Mhg-FLCZ using a binocular microscope (Labomed LX-300).Using binocular microscopy at different magnification ranges as 1000x and 1500x to analyze the morphology and size uniformity of Mhg-FLCZ [21,22].

In vitro release profile of Mhg-FLCZ
The Franz diffusion cell instrument (Orchid Scientific EMFDC-06, India) was used to conduct in vitro diffusion investigations [23,24].Dialysis membrane with a molecular weight cut off of 12000 to 14000 Da from Hi-Media Laboratories Pvt. had a receptor compartment capacity of 20 ml and a donor compartment exposure area of 1.41 cm.Dialysis membrane has a flat width of 24.26 mm and a diameter of 14.3 mm with an approximate capacity of 1.61 ml/cm was used for the study.The membrane was soaked overnight in phosphate buffer pH 7.4.10 ml of prepared Mhg-FLCZ (containing phosphate buffer), which contains 10 mg FLCZ was taken and placed in the donor cell.A dialysis membrane was placed in between the donor cell and the receptor cell.20 ml of phosphate buffer (pH 7.4) was taken in the receptor cell to touch the bottom surface of the dialysis membrane.The temperature of the receptor phase was maintained at 37±0.5 °C and the receptor compartment was stirred with a magnetic stirrer to maintain a homogeneous condition.The aliquots of 1 ml were withdrawn at different time intervals.Fresh medium was used to replace an equal volume of the sample withdrawn.The samples were analyzed at 260 nm in a UV-visible spectrophotometer (Shimadzu 1800) and the amount of drug released at different time intervals was calculated.In vitro analyses were performed for 8 h.Numerous kinetic studies, including first order, zero order, Korsmeyer-Peppas model, and Higuchi's model, were carried out to ascertain the manner and mechanism of Mhg-FLCZ release from various formulations.

Ex-vivo permeation study of Mhg-FLCZ
The optimized formulation was tested for permeation in ex-vivo using Franz's diffusion cell (Orchid Scientific EMFDC-06, India), which has a 3.14 cm 2 diffusion area.This research used rat abdominal skin for the permeation.The receptor compartment was pointed towards the dermis, whereas the donor compartment was pointed towards the stratum corneum of the excised rat skin.The donor compartment was filled with Mhg-FLCZ gels, and the receptor compartment was filled with 20 ml of medium [25,26].
On the receptor side of the experiment, Teflon-coated magnetic beads were used to stir the solution at 50 rpm while maintaining it at 37±0.5 °C.Following the injection of the test formulation on the donor side, at predetermined intervals of 1, 2, 4, 6, and 8 h, to maintain the sink condition, 1 ml of samples were taken from the receiver compartment and after each sample was taken, an equal volume of receptor fluid was injected into the receiver compartment.Using a UV-visible spectrophotometer with a maximum wavelength of 260 nm, this study assessed the amount of Mhg-FLCZ gel in receptor fluids [27,28].

Statistical design
As two independent variables, the amounts of carbopol 940 (A) and NaCMC (B) were varied at three levels: low (-1), medium (0), and high (+1).Performance in a factorial design (two factors and three levels) was used to modify the Mhg-FLCZ for best performance.Different levels of independent variables that are taken into consideration during the Mhg-FLCZ optimization process were applied to the trial batch's operation.There are three separate dependent or response variables: entrapment efficiency (%) (Y1), viscosity (cps) (Y2), and percentage (%) of drug release at 8 h (Y3).Throughout the optimization process, these were taken into consideration.The trial version of Design Expert 11 was used to generate and analyze experimental data.The independent and dependent variable designs are shown in table 2.
The Mhg-FLCZ was developed based on the optimization of independent variables on dependent variables by the application of a polynomial equation with parameters such as independent factors and interaction with observed responses, which were taken into consideration in the research [30,31].
In the present evaluation, the dependent variable was Y, the intercept value was b0, the regression coefficients employed were b1, b2, b3, and b5, and the response factors were A and B. AB is thought to represent an interaction between the variables A and B. One-way ANOVA was used to determine the significance of the models (p<0.05) and individual response parameters.Entrapment efficiency (%) (%) (Y1), viscosity (cps) (Y2), and percentage (%) of drug release at 8 h (Y3) were shown to preserve the best response acceptability.

Statistical analysis
With the support of GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA, USA), the data was analyzed and reported as mean (SD).The formulation was optimized using the trial version of Design Expert Software, Version 13.0.A difference below the probability threshold of a P-value of 0.05 was determined using ANOVA.

Formulation optimization
The quadratic model is used to identify how to analyze individual primary components and interaction factors utilizing the design expert program (Design Expert 11, State Ease Inc., USA). 3 2 full-factorial designs were used to optimize the various examined responses, each of which is represented by a quadratic as follows.The amount of CP (A) and amount of NaCMC (B) were utilized as independent variables in this full factorial design based on responses from multiple trial batches and changed at three different levels: low (-1), medium (0), and high (+1).Entrapment efficiency (%), Viscosity (cps), and percentage (%) of drug release at 8 h are the three separate response variables.The quadratic model was used to identify how to analyze individual primary components and interaction factors using the design expert software (Design Expert 11, Stat Ease Inc., USA).Using a quadratic equation, the various explored responses are indicated as follows.The high correlation coefficient (R 2 ) value from the aforementioned model equations shows that the variables under investigation have a significant influence on the investigated responses.The ANOVA results (table 3) clearly showed that both factors-the quantity of CP and the percentage of w/v NaCMC-had significant (p<0.05)impacts on drug release at 8 h (%), viscosity (cps), and entrapment efficiency (%).
Eliminating the non-significant (p>0.05)components allowed for model reduction of the above-mentioned quadratic equations.
Fig. 2A shows linear correlation graphs comparing the actual and expected findings, as well as residual plots linking entrapment efficiency (%), viscosity (cps), and drugs released at 8 h (%).The corresponding residual plots showing the scatter of the observed versus predicted values relating to entrapment efficiency (%), viscosity (cps), and drug released at 8 h (%).are presented in fig. 2 (A), 2 (B) and 2 (C), respectively.The influence of both independent variables (The amount of CP and % w/v of NaCMC) on investigating responses (entrapment efficiency (%), viscosity (cps), drug released at 8 h (%) was further explicated using two-dimensional contour plots and three-dimensional response surface plots.
The contour plot relating entrapment efficiency in fig. 2 (D) depicted the non-linear decrease of entrapment efficiency (%) with an increase in % w/v of NaCMC.However, the entrapment efficiency (%) was linearly increased with an increase in the amount of the amount of CP (A).At low and high levels of B, entrapment efficiency was significantly (p<0.05)increased when the amount of CP (A) increased from −1 level to+1 level.The response surface plot relating entrapment efficiency (%) (fig.3  (C)) indicated that the entrapment efficiency (%) was more dependent upon the amount of CP (A) compared to the % w/v of NaCMC (B).The response surface plot also showed the strip-linear curvature on the axis of the amount of CP (A), while an almost flattering curve was seen for the % w/v of NaCMC (B).

Fig. 3: (A) Contour plot demonstrating the impact of the amount of CP (A) and % w/v NaCMC (B) ratio on the Mhg-FLCZ viscosity. (B) Contour plot demonstrating the impact of the amount of CP (A) and % w/v NaCMC (B) on the ratio of the Mhg-FLCZ drug release at 8 h. (C) Response Surface plot demonstrating the amount of CP (A) and % w/v NaCMC (B) ratio on the entrapment efficiency (%) of Mhg-FLCZ. (D) Response Surface plot demonstrating the amount of CP (A) and % w/v NaCMC (B) ratio on the viscosity (cps) of Mhg-FLCZ. (E) Response Surface plot demonstrating the amount of CP (A) and % w/v NaCMC (B) ratio on the % drug release at 8 h of Mhg-FLCZ
The contour plot illustrates the viscosity (cps) in fig. 3  The contour plot (fig.3 (B)) of the drug released at 8 h (%) versus factors A and B despite the linear increase of drug released at 8 h with an increase in the amount of % w/v NaCMC (B).On the other hand, a non-linear slight increment of drug released at 8 h (%) was observed with an increased amount of CP (A).At low and high levels of A, the drug released at 8 h (%) was significantly (p<0.05)increased as B was increased from −1 level to+1 level.This designated that the amount of % w/v NaCMC (B) had pronounced effects on the drug released at 8 h (%) as compared to the amount of CP (A).
According to the equation above, a high correlation coefficient value indicates that the experiment's components and the analyzed responses are closely related.According to the ANOVA result, both of the factors, the amount of CP (A) and % w/v NaCMC (B) had a significant effect on drug entrapment efficiency (%), viscosity, and percentage (%) drug release at 8 h, with a p-value of 0.05 or below.
The quadratic model's simplicity was seen as a key concept.

Entrapment efficiency (EE%)
The entrapment efficiency (%) of various Mhg-FLCZ is presented in table 2. entrapment efficiency (%) of Mhg-FLCZ was found within the range of 36.08±0.32% to 65.09±0.41%.The poor entrapment efficiency (%) of Mhg-FLCZ might be low solubility and poor lipophilicity.It has been reported that the microparticles are capable of reducing drug leakage, prolonging the residence time of the drug on the skin, and facilitating the internalization of drugs into cells.The entrapment efficiency (%) was found significantly (p<0.05)increased as the amount of CP and % w/v of NaCMC were increased.

pH of Mhg-FLCZ
Triethanolamine was used in small quantities in all batches (F1-F9) to maintain the pH close to the skin's (5.6).All batches have pH values ranging from 4.1±0.18 to 5.6±0.28.To prevent skin irritation and other associated issues, Mhg-FLCZ gel formulations should have a pH value adjusted to be close to that of the skin [32].The pH and viscosity of hydrogel thickened microemulsion gradually increased with a decrease in concentration of surfactant co-surfactant mixture in the formulation.

Spreadability
Spreadability tests were performed for all formulations, and for the Mhg-FLCZ gel formulation, the spreadability of the gel formulation reduced as the polymer concentration increased.The spreadability range of the Mhg-FLCZ was found to be 1.28±0.04g. cm/s to 3.6±0.01g. cm/s.Spreadability is the term utilized to describe a cream's ability to spread over the skin.Spreadability is essential because it explains the way gel behaves when it escapes from the tube.As a topical cream gets closer to the skin's surface, its ability to spread decreases [33].FT-TR (Fourier transform infrared) Fig. 5(A) demonstrates that the IR of fluconazole exhibits absorption peaks at 3112.48 cm -1 for stretching of the-OH group, 1510.54 cm -1 for stretching of the triazole ring, 1111.96cm -1 for bending of the C-F chain, 1361.07 cm -1 for bending of the group, 1728.91 cm -1 for of the C=O chain, and 912.28 cm -1 for stretching of the C-C chain.

Viscosity
Fig. 5(B) shows that IR of fluconazole and polymer given a peak at 3113.35 cm -1 , 1510.39 cm -1 , 1111.79 cm -1 , 1361.24 cm -1 , 1729.71 cm -1 , 912.18 cm -1 for-OH stretching, triazole ring stretching, C-F bending,-OH bending of phenyl, C=O bending, and C-C stretching respectively.As a result of the stretching and bending of all peaks, it was determined that there was no molecular interaction between the drug and polymers because of the persistence of their internal structural and geometric configurations.

Kinetic analysis of release data
The Mhg-FLCZ formulations' in vitro FLCZ release data has been represented using a variety of kinetic models.Table 5 displays the correlation coefficient (R 2 ) value after release data was fitted to multiple kinetic models and release processes.According to the highest correlation coefficient (R 2 ), the Higuchi equation (0.9762) was followed by zero-order kinetics (0.9858) in the release patterns of F2 formulation Mhg-FLCZ.

Optimization of Mhg-FLCZ by 3 2 factorial design
The polynomial equations for the independent and response variables were used to optimize for all three answers in order to find the best formulation.After trading off several answer factors, the following acceptable response ranges have to be limited: Viscosity (cps) is 11100±1.21;entrapment efficiency (%) is 65.09±0.41%;and drug release at 8 h (%) was 93.22%.It was determined, by a thorough analysis of the possibility search and subsequent grid studies, that the formulation comprising 1.5% w/v of CP and 1.5% w/v of NaCMC satisfies all requirements.
The produced Mhg-FLCZ formulation of F2 had an 8-hour drug release of 93.22% and an entrapment efficiency of 65.09±0.41%.Based on the aforementioned results, formulation F2 was found to be an optimized formulation.By using design expert trial version 13 software, optimized Mhg-FLCZ was selected for the F2 formulation because its lack of error (22.89) for the response of the dependent variables was the lowest of all comparisons with other formulations.

Fig. 2 :
Fig. 2: (A) An analysis of % Entrapment efficiency between actual and predicted values is illustrated in this linear correlation plot.(B) A linear correlation plot is shown between the actual and predicted viscosity of Mhg-FLCZ.(C) Comparison of the actual and predicted release of % of drug at 8 h shown in a linear correlation plot.(D) Contour plot demonstrating the amount of CP (A) and % w/v NaCMC (B) affect the percentage of entrapment efficiency (%) (A).The amount of CP and B (% w/v of NaCMC) showed a linear increase in entrapment efficiency (%) with an increase in the amount of CP (A) and %W/V of NaCMC (B).At a low level (−1) of % w/v of NaCMC (B), the viscosity (cps) was significantly (p<0.05)increased when the amount of CP (A) increased from −1 level to+1 level.Moreover, a similar result was observed on the viscosity (cps), A was increased from −1 level to+1 level at a high level of B (+1).The response surface plot relating viscosity (cps) (fig.3 (D)) designated the slightly linear curvature on the axis of the amount of CP (A) and %w/v of NaCMC (B).This indicated that both the amount of CP (A) and % w/v NaCMC (B) had a considerable effect on viscosity (cps).

Fig. 8 :
Fig. 8: (A) Graph of the linear relationship that exists between the actual and expected entrapment efficiency (%).(B) Graph of the linear relationship that exists between the actual and expected to viscosity (cps).(C) Graph of the linear relationship that exists between the actual and expected values for drug release at 8 h (%)