**Development and Evaluation of Polymeric Nanosponge Hydrogel for Terbinafine Hydrochloride: Statistical Optimization, In Vitro and In Vivo Studies**

**Aditee Ghose 1,**†**, Bushra Nabi 1,**†**, Saleha Rehman 1, Shadab Md 2,3 , Nabil A. Alhakamy 2,3 , Osama A. A. Ahmad 2,3 , Sanjula Baboota <sup>1</sup> and Javed Ali 1,\***


Received: 14 October 2020; Accepted: 1 December 2020; Published: 3 December 2020

**Abstract:** Terbinafine hydrochloride, although one of the prominent antifungal agents, suffers from low drug permeation owing to its hydrophobic nature. The approach of nanosponge formulation may thus help to resolve this concern. Thus, the present research was envisioned to fabricate the nanosponge hydrogel of terbinafine hydrochloride for topical delivery since nanosponge augments the skin retentivity of the drug. The optimized formulation was obtained using Box Behnken Design. The dependent and independent process parameters were also determined wherein polyvinyl alcohol (%), ethylcellulose (%), and tween 80 (%) were taken as independent process parameters and particle size, polydispersity index (PDI), and entrapment efficiency (EE) were the dependent parameters. The nanosponge was then incorporated into the hydrogel and characterized. In-vitro drug release from the hydrogel was 90.20 ± 0.1% which was higher than the drug suspension and marketed formulation. In vitro permeation potential of the developed formulation through rat skin showed a flux of 0.594 <sup>±</sup> 0.22 <sup>μ</sup>g/cm2/h while the permeability coefficient was 0.059 <sup>±</sup> 0.022 cm/s. Nanosponge hydrogel was evaluated for non-irritancy and antifungal activity against *C. albicans* and *T. rubrum* confirming the substantial outcome. Tape stripping studies exhibited ten times stripping off the skin quantified 85.6 <sup>±</sup> 0.21 <sup>μ</sup>g/cm2. The confocal analysis justified the permeation potential of the prepared hydrogel. The mean erythemal score was 0.0, confirming that the prepared hydrogel did not cause erythema or oedema. Therefore, based on results obtained, nanosponge hydrogel formulation is a potential carrier for efficient topical delivery of terbinafine hydrochloride.

**Keywords:** nanosponge; hydrogel; Box–Behnken design; pharmacokinetic; terbinafine hydrogel

#### **1. Introduction**

Fungal infections currently account for about the fourth common disease in the world that affects millions of people every year, about 25% of the world's population, mostly adults. The enhanced manifestation of the infections has led to increased cases of immunocompromised patients suffering from malignancies, HIV, and diseases of the similar sought [1–3].

The skin serves as a defence system for the human body when exposed to ultraviolet radiation that induces oxidative stress. Oxidative stress may lead to lipid peroxidation and DNA breakage on some occasion. However, there are certain self-defence mechanisms, such as superoxide dismutase, available for protection [4,5]. Furthermore, in the current scenario, skin and specifically stratum corneum are commonly used for drug delivery. The stratum corneum is considered as the target organ for the delivery of antimycotic drugs because the desirous concentration of drugs to render the therapeutic effect could be easily achieved post topical delivery. However, to achieve the same peroral effect, a higher dose is needed, resulting in a higher incidence of adverse effects [6,7].

Topical drug delivery systems have established a reputation for themselves and have the potential for efficient drug delivery due to the vehicles used in their preparation that ultimately affect the rate of drug permeation across the skin [6]. Topical delivery provides great advantages such as patient compliance due to ease of applicability and non-invasive design, on-site delivery minimizing systemic side effects and effective targeting ability. Certain drawbacks are associated with the conventional formulations. Therefore, to overcome the lacunas, hydrogels are tailored and widely used for topical application. The fact that they are widely gaining impetus is attributable to their swelling property in association with their adhesiveness and potential to modulate the drug release [8].

Nanosponges (NS) are a novel formulation, a sponge-like structure used to encapsulate nanoparticles with a non-collapsible and porous structure. It is primarily used for pharmaceutical and cosmeceutical approaches, as it blends the advantages of microsponges and nanosized vesicular structure. The porous structure not only enables us to entrap a wide range of active ingredients but also modulates the release pattern. NS, if incorporated in hydrogel offers remarkable perks, the most important being improved skin retention [9,10]. NS offers remarkable advantages including higher entrapment efficiency, improving the drug profile, economical method of preparation, and ease of drug release owing to three-dimensional porous structures. Different preparation methods are used: solvent method, ultra-assisted synthesis, emulsion solvent diffusion method, and melting method to formulate stable NS in different categories. Software-based optimization techniques are employed to derive optimized product of superior attributes and quality. Moreover, 3D printing techniques are now being considered to ease the production of NS. Different routes and modes of drug administration e.g., aerosols, capsules, parenteral, tablets, topicals are now being exhausted for NS delivery [11].

Terbinafine hydrochloride (TH) is the fungicidal allylamine drug that kills the fungal organisms. It acts as a non-competitive inhibitor of squalene epoxidase lodging within the fungi cell membrane. Furthermore, it plays an active role in diminishing the level of ergosterol along with the lowering of the intracellular buildup of squalene which leads to fungal cell death. Fungi often infect the skin surface and eventually invade the stratum corneum, and desquamation from the skin surface is not possible [12]. The drug is hydrophobic thus exhibiting poor drug permeation. Hence to overcome the drawbacks of the conventional topical formulations, a novel approach has been envisaged to demonstrated promising results. Cerebi et al. and Vagashiya et al. developed TH microemulsion-based hydrogel and solid lipid nanoparticles, which were instrumental in reducing the fungal load [8,13]. Barot and colleagues developed the microemulsion of TH. The cumulative amount of drug permeation was enhanced 3-folds when compared with conventionally available cream [14]. Mahaparale and colleagues fabricated TH polymeric microsponge using a quasi emulsion solvent diffusion method. The studies established that the sustained release mechanism was followed by the developed formulation. Satisfactory drug deposition was also deduced [15]. Amer et al. showed the importance of developing TH nanosponge that exhibited 90% of drug release within 8 h. Furthermore, the highest in vivo skin deposition and antifungal activity were also demonstrated in the developed formulation [16]. However, the study conducted on NS of TH was not exhaustive enough and lacked an in-depth analysis and discussion section. Thus, the authors intended to conduct extensive analysis, thereby, providing a novelty quotient to the research envisaged.

The purpose of the research undertaken is to formulate and optimize NS of TH and further incorporate it into the hydrogel. This step would be superseded by the in vitro release and permeation studies followed by an antifungal activity. This aids in determining the in vivo prospect of TH.

#### **2. Experimental Methodologies**

#### *2.1. Materials and Animals Used*

#### 2.1.1. Materials

Terbinafine Hydrochloride (TH) was received from A.S Lifesciences, (Haryana, India). Other ingredients used were Polyvinyl Alcohol (PVA) (GS Chemical Testing & Chemical Industries, New Delhi, India); Ethyl Cellulose (EC) (Titan biotech Ltd., Rajasthan, India); Dichloromethane, Tween 80, Carbopol 940, and Ethanol (S.D. Fine Chemicals Ltd., Mumbai, India); Triethanolamine (Loba Chemie, Mumbai; India). Analytical grade reagents were used for the research.

#### 2.1.2. Animals

Albino Wistar rats (weighing 200–250 g) were chosen as the animal model which was obtained by Central Animal House, Jamia Hamdard, New Delhi upon approval from the IAEC, Jamia Hamdard (173/GO/Re/S/2000/CPCSEA)(Protocol Number: 1404) and CPCSEA (Registration number 173/CPCSEA), Government of India.

#### *2.2. Development and Characterization of TH-NS*

#### 2.2.1. Optimization of TH-NS

Significant endeavours were made for the fabrication of the optimal NS for which the quality target product profile (QTPP) was determined. This step was instrumental in establishing the plausible attributes of the end product. Thus, it is a vital stage that needs to be emphasized to deduce the finished product of high standards concerning quality, safety, and efficacy. Nevertheless, this could be accomplished only by focusing on critical process parameters (CPP) and critical material attributes (CMA) that might, in turn, affect the CQA (critical quality attributes) [17]. The different essentials of QTPP with their validation for the fabrication of TH-NS are mentioned in Table 1. These QTPP have specific targets that need to be addressed for the research. For instance, the product developed should have optimal features to allow the route of administration to be topical. The Ishikawa plot as represented in Figure 1 elaborates on the various risk factors, conditions required, along with CQA in the development process.


**Table 1.** QTPP with its target and justification for the fabrication of TH-NS.

**Figure 1.** Ishikawa diagram used as a visualization tool for the fabrication of TH-NS.

#### 2.2.2. Fabrication of TH-NS

The NS was developed by the emulsion diffusion method using different proportions of excipients i.e., EC, PVA, and Tween-80. The continuous phase comprised of 17.5 mL aqueous solution of PVA in different ratios, with varying amounts of surfactant, and the dispersed phase consisting of TH (100 mg) and in 2 mL of dichloromethane optimum quantity of EC is dissolved. The organic phase was incorporated in the aqueous phase maintaining the requisite conditions (temperature: 35 ◦C, 1000 rpm for 2 h). The resultant NS was collected and dried at 40 ◦C for 24 h [18].

The optimization process was carried out using a Box–Behnken design wherein PVA (%), EC (%), and Tween 80 (%) were taken as independent process parameters. The dependent parameters selected for the optimization process were particle size, polydispersity index (PDI), and entrapment efficiency (EE) [19]. The relation between variables and expected goals is depicted in Table 2. The various independent and dependent variables are demonstrated in Table 2. The levels of independent variables are also mentioned. The three-factor 3-level design employed led to 17 experimental runs, yielding a quadratic equation defining the model, as shown in the equation below.

$$Y = b\_0 + b\_1 \mathbf{x}\_1 + b\_2 \mathbf{x}\_2 + b\_3 \mathbf{x}\_3 + b\_4 \mathbf{x}\_4 + b\_{12} \mathbf{x}\_1 \mathbf{x}\_2 + b\_{13} \mathbf{x}\_1 \mathbf{x}\_3 + b\_{23} \mathbf{x}\_2 \mathbf{x}\_3 + b\_{11} \mathbf{x}\_1^2 + b\_{22} \mathbf{x}\_2^2 + b\_{33} \mathbf{x}\_3^2 \tag{1}$$

**Table 2.** Dependent and independent variables of Box-Behnken Design with their respective levels and goals.


*Y*: effect observed from the combination of different levels of process parameters; *b*0: intercept, *b*1–*b*3: regression coefficients; *x*1–*x*3: independent variables taken for study; *b*12, *b*13, *b*23: interaction coefficients; *b*11, *b*22, *b*33: quadratic coefficients of the experiment conducted.

#### 2.2.3. Characterization of Prepared TH-NS

Particle size, polydispersity index (PDI), and entrapment efficiency (EE).

The particle size and PDI were measured using Malvern zeta sizer (Malvern Instruments Ltd., Worcestershire, UK). Sample dilution was performed using distilled water at 25 ◦C to yield dispersion of sufficient scattering intensity, and analysis was performed at 372.0 (kcps) for 20 s.

TH-NS was weighted and centrifuged at 10,000 rpm for 30 min. It was diluted in 10 mL methanol. This step is envisaged to break the aggregates if formed. The mixture was sonicated for 5 min. It was filtered and analyzed at 223 nm by UV spectrophotometric technique [1].

> % Drug entrapmentefficiency <sup>=</sup> <sup>W</sup>(encapsulatedNS) <sup>×</sup> <sup>100</sup> W(totaldrug)

Again, the particle size determination was further validated using transmission electron microscopy (TEM). The sample for analysis was diluted 10 folds. It was then placed on the copper grid immersed within a copper film. This step was then followed by staining with phosphotungstic acid (1%) to provide a negative charge. Finally, the preparation was air-dried before viewing [20].

The pure drug and TH-NS (5 mg each) were utilized for carrying out DSC analysis wherein Perkin Elmer differential scanning calorimeter was used. The requisite conditions were maintained (heating rate: 10 ◦C/min, temperature: 30–400 ◦C, and nitrogen flow: 60 mL/min) [20].

#### *2.3. Fabrication of Topical Hydrogel Integrating TH-NS*

The TH-NS was dispersed in 1% *w*/*w* Carbopol 940 to fabricate hydrogel (HG). Various TH concentrations were measured at 0.2, 0.5, 0.8, 1 and 1.5% *w*/*w*. The 2-h polymer dispersion in water allowed it to act as a gel-forming agent. The external force was employed by agitating the established system at 600 rpm, followed by a 15-min stagnation process. This move is critical in removing trapped air. Additionally, 2% *v*/*v* of aqueous triethanolamine was added to the prepared formulation by holding it at 100 rpm.

#### *2.4. Characterisation of TH-NS HG*

#### 2.4.1. Visual Observation

The prepared HG was inspected visually for its colour, homogeneity, grittiness, and syneresis [21].

#### 2.4.2. Viscosity Determination

The prepared gel viscosity was determined using a programmable Viscometer. The HG prepared was taken in a beaker wherein the T-bar spindle (spindle-C, S-96) was immersed at 90◦ such that the spindle and the base of the beaker are not in contact. The speed of rotation of the spindle was maintained at 50 rpm. The evaluation is made after a duration of 30 s post which the HG system prepared is stabilized [12,22].

#### 2.4.3. pH Determination

The pH meter was used to determine the formulation pH. In a known quantity of distilled water (100 mL), the prepared HG (1 g) was dissolved and kept for 2 h. The electrode was then immersed in the mixture produced and observed at room temperature in triplicate [21].

#### 2.4.4. Spreadability

Spreadability (g·cm/s) is referred to the time in sec required by two slides to slip over the HG placed between them upon the influence of external stimuli. The glass slides of 7.5 cm in length were employed along with the load on the upper plate being 20 g [23]. Spreadability was evaluated using the formula below mentioned:

$$\text{Spreadability} = \frac{\text{Weight (g)} \times \text{Length (cm)}}{\text{Time (s)}}$$

#### 2.4.5. Texture Analysis

The mechanical property of HG was evaluated with the help of software-texture analyzer TA (XT *Plus* Stable Micro System, UK equipped with 5 kg load cell). The sample weighing 100 mg was placed in the beaker cautiously to avoid air bubbles in it. The speed of the probe for analysis was fixed at 1.0 mm/s for pre-test, 2.0 mm/s for the test, and 10.0 mm/s for post-test analysis.

The probe was immersed with a load cell capacity of 5.0 g and a distance of 10.0 mm. Data interpretation was undertaken using the Texture Exponent software installed within the equipment. The resultant force-time plot gave the values of different mechanical parameters [24,25].

#### 2.4.6. Determination of Drug Content

The prepared HG (50 mg) was dissolved in 100 mL phosphate buffer pH 5.5 and shaken for 2 h. This move is intended to ensure optimum drug solubility during mechanical shaking. The solution was purified and spectrophotometrically quantified at 223 nm [6,21].

#### *2.5. In Vitro Release Study*

The in vitro drug release study of TH from the drug suspension (DS), TH-NS, HG, and the marketed formulation was determined using an activated dialysis bag in a dissolution chamber where the system was maintained at 100 rpm and 37 ± 0.5 ◦C. The release profile was determined in phosphate buffer pH 5.5 (pH of normal skin) and phosphate buffer pH 7.4 (physiological pH) respectively. Sample equivalent to 3 mL was removed at specified time intervals and an equal quantity of fresh dissolution media was replenished in the assembly to maintain sink condition. Filtered and quantitatively determined at 223 nm. The data obtained were fitted to different kinetic models to interpret drug release mechanisms and kinetics [12,26].

#### *2.6. In Vitro Permeation Studies*

The rat's skin was excised, washed using distilled water before mounting on the Franz diffusion cell. The skin was so positioned that the donor compartment faced the stratum corneum (SC) and the dermis side faced the receptor. The HG was taken as necessary and put on the skin surface. The receptor compartment consisted of phosphate buffer pH 5.5, taken as the diffusion media. The appropriate condition was maintained at 37 ± 1 ◦C along with constant aeration. Sample (1 mL) was removed at a predetermined time and reloaded with fresh media. The cumulative amount of drug permeation as a function of time along with flux (J) was statistically determined using HPLC analysis [1,6,27].

#### *2.7. Confocal Laser Microscopy*

Confocal microscopic examination is essential to the drug's in vivo prospects. The HG prepared by labelling it with Rhodamine 123 dye, applied to Albino Wistar rats' dorsal skin and allowed for 8 h. Then the animal was slaughtered, skin excised and washed with phosphate buffer pH 5.5. Prepared skin section was placed on a slide and analyzed microscopically using CLSM (Leica microsystems). For rhodamine 123 operation, optical excitation was performed with a laser beam of 488 nm and fluorescence emission was detected above 560 nm [22,28].

#### *2.8. Tape Stripping Technique*

Post conducting the in vitro permeation investigation, the surplus quantity of the developed formulation was scrapped off from the skin surface. It was then washed thrice with the phosphate buffer (pH 5.5) and dried gently using a cotton swab. The final process envisaged the use of a serial tape-stripping method whereby, the removal of 15 strips using adhesive tape was undertaken. Each tape was carefully weighed before and after the procedure. The adhesive tapes were collected using a methanol-water mixture (95:5 *v*/*v*) and further analyzed using the HPLC technique [28,29].

#### *2.9. Skin Irritation Study*

The test of skin irritation potential was carried out with the HG prepared in comparison to placebo HG and performed by the Draize patch test. The animals were divided into the following groups (*n* = 3)


The animal's dorsal side was shaved 24 h before drug application. HG (0.5 g) was applied evenly and homogenously to hair-free rat skin covering 4 cm2. Any visual improvement was monitored on rat skin after 24, 48, and 72 h post HG applicability. The mean erythema scores of the different formulations were graded from 0 to 4 [10,30].

#### *2.10. Antifungal Activity Study (Cylinder Plate Method)*

*Trichophyton rubrum* and *Candida albicans* species were used to determine HG antifungal inhibitory activity. For the analysis of antifungal potential, the cylinder plate method was selected using sabouraud dextrose media (Himedia, India). The requisite conditions are 25 ◦C for 7 days and 30 ◦C for 2 days for *Trichophyton rubrum* and *Candida albicans* respectively. The spores were then harvested and suspended into the media of amount 20 mL. The inoculated media (1 mL) was incorporated into 100 mL of sabouraud dextrose agar at 37 ± 1 ◦C. From the mixture thus obtained, 10 mL was inserted in the petri dish in solidified agar media. Four wells were bored in a media-containing petri dish and filled with 0.1 g HG, placebo HG, advertised formulation and control (distilled water). Different Petri dishes contained different microbial strains from which zone of inhibition was determined using a hemocytometer [6,14].

#### *2.11. Statistical Analysis*

The results obtained in different studies were statistically analyzed. The experiments were conducted in triplicate and expressed in the form of mean ± SD. One-way analysis of variance (ANOVA) (GraphPad Software Inc., San Diego, CA, USA) was employed for the determination.

#### **3. Results and Discussion**

#### *3.1. Optimization of TH-NS*

The primary target was to tailor TH-NS with enhanced skin retentivity. The prime concept behind predefining the QTPP was to get a patient-centric formulation of the utmost quality that would offer maximum therapeutic outcome. Therefore, the attributes, i.e., particle size, EE, and PDI, were selected as CQA which offers its respective advantages towards the developed systems.

The determination of process parameters in the optimization process is of prime concern to yield effective and economical outcome. The conventional "one-factor-at-a-time approach" is time-consuming along with avoidance of interactions between independent variables, thereby paving way for novel optimization techniques. Response surface methodology (RSM) is a potential optimization tool wherein, multiple factors and their interactions could be ascertained with a minimal number of experimental runs. In this method, the response of interactions of statistically designed combinations coupled with the determination of the coefficients of the best fit model has endeavoured to predict the adequacy of the model [31]. The outcome of the trails was accessed using Design-Expert software which validated the substantial use of statistical design methodology. The independent process attributes chosen were PVA, EC concentration, and Tween80 concentration for which the dependent parameters were particle size (nm), PDI, and EE (%). Polynomial quadratic equations along with respective correlations were derived based on the interactions of the dependent and independent variables. The contour plots thereby obtained from the analysis demonstrated the qualitative effect of each variable on each response.

On applying the design, 17 runs were obtained. The result obtained is shown in Table 3 and the contour plots are shown in Figure 2. The result obtained from the experimental runs as evident from Table 3 reveals that the particle size of the different formulations was in the range of 425.7–571.4 nm. The PDI was in the range of 0.30–0.564 while EE lies between 50.5% and 85.45%.


**Table 3.** Experimental runs of BBD with obtained results.

**Figure 2.** (**A**) Contour plot depicting size based on the concentration of PVA and EC% (**B**) Contour plot depicting PDI based on the concentration of PVA and EC% (**C**) Contour plot depicting entrapment efficiency based on the concentration of PVA and EC%.

The second-order polynomial equation relating the response of particle size (Y1), PDI (Y2), and EE% (Y3) respectively are given below:

Y1 = 28.94B + 19.66C − 40.80AB + 29.51BC Y2 = 0.0060A − 0.0340B + 0.0570C + 0.0262AB + 0.0502AC + 0.0487BC

#### Y3 = 71.96A + 12.14B + 2.87C

The positive sign in the quadratic relation depicts a synergism while on the contrary, the negative sign shows an antagonistic relation. A backward elimination process was adopted to allow the data to fit within the quadratic model obtained. It could be observed that PVA concentration had a major effect on particle size followed by PDI while the minimum effect on EE [10].

#### *3.2. Characterisation of Optimised TH-NS*

#### 3.2.1. Particle Size Determination, Polydispersity Index, Entrapment Efficiency

The particle size of prepared TH-NS was 448.4 ± 12.6 nm, while PDI was 0.3 ± 0.04. The findings observed were based on earlier literature [12]. The entrapment efficiency of the TH-NS was found to be 85.45 ± 3.7% which was further corroborated from the given literature of loaded NS [10,18]. TEM image showed that the prepared TH-NS morphology was spherical with a size of 440 nm as shown in Figure 3. The result obtained was consistent with the zeta sizer analysis [10,12].

**Figure 3.** TEM image of TH-NS.

#### 3.2.2. Thermoanalytical Technique (DSC)

Pure TH gives a sharp endothermic peak at 214 ◦C evaluated by DSC spectra as depicted in Figure 4. However, when the DSC spectra of TH loaded NS formulated was compared with spectra of pure drug, it showed the difference in absence of peak formations at the temperature as depicted in Figure 4. The absence of peak demonstrated that the TH loaded NS has been developed which leads to the reduction in crystallinity of the drug. The change in the structure from crystalline to amorphous established the fact that NS has been developed [1]. The molecularly dispersed phase of TH within the NS structure leads to the broadening of the peak. The process seems to be following the Gibbs-Thompson equation [32]. The amorphous structure of the TH-NS established by the result obtained is desirable for enhanced drug entrapment within the NS structure [33].

**Figure 4.** DSC thermogram of pure drug and TH-NS.

#### *3.3. Preparation and Evaluation of TH-NS HG*

Preliminary tests were conducted to determine the best formulation to prepare the optimum gel. Table 4 indicates a different HG composition. The Carbopol 90 composition ranged from 0.2% to 1.5% *w*/*w*. However, the compositions of other constituents remained constant at a fixed concentration. The effects of the compositions on the following parameters were tested. These properties are instrumental in establishing the physical stability of the prepared hydrogel [8]. The purpose of following such objectives was to optimize the formulation on the basis of experimental demonstrations. Table 5 demonstrates the results obtained for different HG preparations.


**Table 4.** Composition amount of HG preparation.



*Polymers* **2020**, *12*, 2903

#### 3.3.1. Visual Examination

The prepared HGs were white, uniform, and clear homogeneity, missing lumps and syneresis, and showed no signs of gritty. The HGs had a glossy appearance except for the G6 formulation showing lumps in it. G5 s formulation was also slightly clumpy. Table 5 shows the result.

#### 3.3.2. Viscosity

Viscosity was testified as an important rheological physical parameter for topical formulations, whichever affects the rate of drug release into the skin. The prepared hydrogels had a viscosity of 0.84–2.99 centipoise. The lowest viscosity was observed in a lower polymer containing hydrogel. The shear rate increases with the decrease in viscosity suggesting the shear-thinning pseudoplastic nature of the formulation. The finding was corroborated with previous literature [6].

#### 3.3.3. pH Determination

The pH value of the prepared HG was found to be 5–6, i.e., within the appropriate limits, and does not cause skin irritation upon application. Additionally, the pH values of different formulations did not change significantly in time.

#### 3.3.4. Spreadability

Spreadability is the degree to which a gel spreads upon application. It was founded that therapeutic effectiveness depends largely on any hydrogel's spreading value. The result showed spreadability within 0.4–2.0 g·cm/sec. Good spreadability is a requirement for ideal gel formulation. The result verified that increased polymer concentration reduces HG's spreadability.

#### 3.3.5. Texture Analysis

Texture can be considered as an index for the product's rheological attribute. For topical delivery systems, the key criteria controlling therapeutic outcome are minimum firmness and maximum adhesiveness. Firmness addresses the product removal from the container and ease of applicability while, on the contrary, adhesiveness pertains to bioadhesion. Hardness was found to lie between 198.10–286.66, adhesiveness between 210.233–286.66, elasticity between 0.372–0.824 and cohesiveness between 0.563–0.802. The results obtained validate that the formed HG has the property of adhering firmly but gently to the skin surface because they have low firmness and high adhesivity.

#### 3.3.6. Drug Content

The percentage of drug content for the prepared HGs were ranging from 82% to 86.5%. Thus, indicating the drug was uniformly distributed throughout the gel.

According to previous studies, the hardness and compressibility of prepared gel should be strong should be low. Furthermore, high cohesiveness is a desirous attribute. Therefore, based on the results obtained from the above-performed studies it could be concluded that G4 with 1% Carbopol is the optimized formulation.

#### *3.4. In Vitro Release Study*

The cumulative % drug release was found to be 83.92% ± 0.22%, 63.06% ± 0.2%, 90.20% ± 0.1%, and 82.83% ± 0.29% respectively in DS, TH-NS, HG, and marketed formulation (Phyte gel) at pH 5.5 (as shown in Figure 5) The difference between the cumulative % drug release in DS and the marketed formulation was not found to be statistically significant. However, statistical significance (*p* < 0.001) was observed when the result obtained for TH-NS was compared with that of DS, HG, and marketed formulation. It was evident that Korsmeyer Peppas kinetic model was followed with R<sup>2</sup> 0.995, 0.998, 0.998, and 0.989 respectively indicating an anomalous diffusion. The result could be due to the matrix's relatively slow diffusion of the trapped drug. It was apparent that HG showed up superior sustained

release potential. As hydrophilic polyacrylic acid polymer, i.e., Carbopol 940 has carboxyl functional groups that get ionized after reaction with TEA. This develops a gel-like structure attributable to the electrostatic repulsion among charged polymer chains. This step leads to an increase in the pH of the HG developed rendering it appropriate for skin applicability. There was no time lag observed in the drug release from the gel observed. The result obtained was following the previously established literature [8,26].

**Figure 5.** % cumulative in vitro drug release.

#### *3.5. In Vitro Permeation Studies*

Permeation study is the passive diffusional study where the drug permeates the entire excised skin (Moreno et al., 2019). The Flux (μg/cm2/h) of DS, HG and marketed gel was found to be 0.10 <sup>±</sup> 0.12, 0.594 ± 0.22 and 0.334 ± 0.23 while permeability coefficient (cm/s) was 0.01 ± 0.012, 0.059 ± 0.022 and 0.033 ± 0.023 respectively. There was statistical significance (*p* < 0.05) when the flux and permeability coefficient of HG was compared with that of DS and marketed formulation. The permeation curve was non-linear followed by a controlled release profile (Figure 6). However, an initial faster release where approximately 20% of the drug was released from the HG in the first 2 h of the experiment. The result was consistent with Malakar and colleagues' analysis [34]. There was an enhanced drug permeation compared to DS and marketed gel due to TH-NS encapsulation in HG. Another related reason for retaining HG on the skin surface may be TH's gradual release pattern over time. Additionally, due to the loose gel structure, the drug release was increased [6,18,35].

**Figure 6.** Cumulative amount of drug permeated vs. time profile.

#### *3.6. Confocal Study*

CLSM analysis was used to confirm HG's permeation potential and drug deposition on the skin for topical application. The skin was examined microscopically after the in vitro permeation of HG and the control solutions. Figure 7 demonstrates the CLSM photomicrographs where Figure 7A is the photomicrographs of cross-sections of hairless viable rat skin, sectioned 0.5 mm. (as standard for comparison), Figure 7B is the skin treated with HG, and Figure 7C is the skin treated with marketed gel 1%. The skin surface was referred to as the brightest autofluorescence of the imaging plane with characteristic morphology of the SC surface. In the images of the SC surface, the corneocyte groups form distinct "island-like" structures forming dark furrows. Increased fluorescence intensity was exhibited by HG. Furthermore, the fluorescence persisted on the upper layers of the skin while diminished rapidly with the depth. The increased fluorescence on the upper layer of the skin due to the retention of hydrogels. It was established that the dye probe distribution was dramatically increased in the topmost dermal layer from the prepared formulations. This might be due to the matrix-forming potential of NS upon integration in hydrogel and probable fusion with the skin membrane [36].

#### *3.7. Tape Stripping Study*

The tape stripping method is a dermatokinetic method to evaluate the distribution of formulation on the skin and in the stratum corneum segregating them into different layers when stripped off. As reported by Morena and colleagues, the concentration of drugs decreases from upper to lower layers. Furthermore, in the tape stripping method, there were chances of the drug being accumulated on the subsequent layers upon an administration which was stripped off leading to a decrease in the drug concentration in the subsequent layers [37,38].

On conducting the method of tape stripping for 10 times after 12 h, it was observed that HG collected in tape stripping was highest 85.6 <sup>±</sup> 0.21 <sup>μ</sup>g/cm2. In the case of TH-NS and marketed gel, it was 6.54 <sup>±</sup> 0.42 <sup>μ</sup>g/cm2 and 24.83 <sup>±</sup> 0.1 <sup>μ</sup>g/cm<sup>2</sup> respectively. Further by 15 times stripping observed HG was reduced to amount 20.64 <sup>±</sup> 0.32 <sup>μ</sup>g/cm2 in comparison to 10 times while TH-NS and marketed were 2.75 <sup>±</sup> 0.31 <sup>μ</sup>g/cm<sup>2</sup> and 9.89 <sup>±</sup> 0.18 <sup>μ</sup>g/cm2 respectively (Figure 8A). The result obtained for HG in the tape stripping method was statistically significant (*p* < 0.001) when compared with TH-NS and marketed formulation after 10 times stripping. However, after 15 times stripping, the statistical significance (*p* < 0.05) was observed when the HG result was compared to TH-NS and marketed formulation (Figure 8A). The outcome obtained further validated that enhanced drug level could be achieved on the subcutaneous layer with lesser penetration into deeper skin layers from the HG developed, a prerequisite for topical drug delivery system. Here, the subcutaneous layer acts as a reservoir to deliver drug progressively to the viable dermis layer. The slower penetration of the developed formulation will also lead to reduced systemic toxicity [33,35]. The study finding that the drug resides on the top of the dermal layer confirmed an enhanced therapeutic drug profile to treat fungal infection [39,40]. The HG demonstrated better results compared to the marketed formulation in both the studies, i.e., the permeation study and the tape stripping study.

**Figure 8.** (**A**) Comparative amount retained by tape stripping (10 and 15 strips) and (**B**) Comparison of the zone of inhibition for the microorganisms of three different groups TH solution, TH-NS gel, and marketed gel.

#### *3.8. Skin Irritation Study*

A control group to which no drug was administered was used in the experiment. The experiment was performed according to the study reported by Iqbal et al., 2018 [41]. The erythemal scores were recorded for all rats at different time intervals of 24, 48, and 72 h. The TH-NS tailored demonstrated the mean erythemal score of 0.00 confirming no erythema or edema on the shaved rat skin, thereby exhibiting minimized skin irritation. However, the drug suspension had a score of 1 at 24 h and post that. As per the degree of erythema, different grades have been allocated no erythema = 0; slight erythema = 1; moderate = 2; more severe = 3; and most severe = 4. The commercially available convention treatments have a considerable drawback as they cause skin irritation, hence the prepared formulation provides an advantage over the conventionally available preparations. The result agreed with the previous literature [3,27].

#### *3.9. Antifungal Studies*

The cylinder plate method was used for the antifungal potential screening wherein the result demonstrated a slight difference in the zone of inhibition between TH-NS, HG, and marketed gel. The diameter of zone of inhibition shown in C. *albicans* and T. *ruburum* were 13 ± 0.42 and 8 ± 0.31 mm by TH-NS, 25.01 ± 0.21 and 22.2 ± 0.32 mm by HG while that from the marketed gel was 22 ± 0.1 and 19.5 ± 0.18 mm respectively. The result obtained for the antifungal study is exhibited in Figure 8B. The result obtained for the zone of inhibition for HG and marketed formulation was found to be statistically insignificant. Although, the result for TH-NS was statistically significant (*p* < 0.05) when compared with HG and the marketed formulation. The HG prepared demonstrated proximity towards the killing of fungal pathogens and demonstrates acceptable susceptibility values against *C. albicans* and *T. ruburum*. Because of the low viscosity of the prepared hydrogel, its penetration was increased compared to the drug solution and marketed formulation [6,42]. Therefore, the TH-NS formulation showed better fungal burden results.

#### **4. Conclusions**

The research undertaken establishes the successful incorporation of TH-NS into hydrogel which was further optimized by the QbD approach (Box–Behnken statistical design). On further in vitro and in vivo study, the prepared formulation provided considerable results, revealing enhanced drug permeability after topical administration. Thus, based on the experimental results obtained, the hydrogel approach is a potential carrier to deliver TH. However, it warrants further elaborate evaluation for commercial applicability.

**Author Contributions:** Conceptualization, A.G., S.B., and J.A.; Methodology, A.G., B.N., and S.R.; Software and Formal Analysis, S.M., O.A.A.A.; Validation, N.A.A. Investigation, J.A. and S.B.; Resources, J.A. and S.M.; Data Curation, B.N. and S.R.; Writing—Original Draft Preparation, B.N., N.A.A. and O.A.A.A.; Writing—Review and Editing, J.A., S.M. and S.B.; Visualization, J.A. and S.B.; Supervision, J.A. and S.B.; Funding Acquisition, S.M. and N.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** The Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, Saudi Arabia has funded this project, under grant no. (FP-134-42).

**Acknowledgments:** The authors are thankful to Jamia Hamdard for providing facilities to carry out the research work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

1. Abdellatif, M.M.; Khalil, I.A.; Khalil, M.A. Sertaconazole nitrate loaded nanovesicular systems for targeting skin fungal infection: In-vitro, ex-vivo and in-vivo evaluation. *Int. J. Pharm.* **2017**, *527*, 1–11. [CrossRef] [PubMed]


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