4.2.2. CVC-Loaded Niosome Optimization through Experimental Design

The Box–Behnken design (BBD) of the Design-Expert software (Stat-Ease Inc.'s, Design Expert®, Version 13) was used to examine the relationships between the three selected input variables (drug, surfactant, cholesterol). The output variables studied were the vesicle size (Y1), polydispersity index (PDI, Y2) and entrapment efficiency (Y3). The input variables were chosen, at their three levels, i.e., their minimum, average, and maximum values, which are shown in Table 8 to explore the final optimal formulation [58].


**Table 8.** Factors and responses employed in BBD to create CVC-Ns.

A, drug (mg); B, surfactant (mg); C, cholesterol (mg); Y1, Vesicle size (nm); Y2, PDI; Y3, EE (%); BBD, Box–Behnken experimental design; Ns, Niosomes; CVC, carvacrol.

### *4.3. Characterization of CVC-Ns*

### 4.3.1. Determination of Globule Size and Zeta Potential

To determine the zeta potential and vesicle size distribution, the dynamic light scattering (DLS) method was utilized. Before analysis, samples were 100-fold diluted in double-distilled water, and impurities were removed using 0.45 m membrane filters. The Malvern particle size analyser equipment (Malvern Instrument Ltd., Malvern, UK) was utilized to determine the zeta potential and to quantify the vesicle size [59].

### 4.3.2. Measurement of % Entrapment Efficiency and % Drug Loading

The drug loading and entrapment efficiency of CVC-loaded niosome vesicles were determined by ultra-centrifuging (Beckman Coulter India Pvt. Ltd., Mumbai, India) formulations for one hour at 15,000 r/min and 4 ◦C. The upper layer of liquid (supernatant) was withdrawn using a pipette, diluted, and filtered (0.25 µm), and the CVC concentration was measured employing UV spectroscopy (UV 1601, Shimadzu, Nagoya, Japan) at 280 nm [60]. The experiment was performed in triplicate. For evaluating the drug-loading capability of the niosomes, drug entrapment efficiency is a crucial factor. This parameter depends on the developmental methodology, the physicochemical features of the drug, and the formulation factors [61].

The equation was used to obtain the entrapment efficiency (EE%) and drug loading (%) [62]:

$$\text{Encapsulation efficiency} \left(\% \right) = \frac{\text{(total amount -- free amount)}}{\text{total amount}} \times 100$$

$$\text{Drug loading } (\%) = \frac{\text{amount of drug entraped}}{\text{total amount of drug and liquid}} \times 100$$

### *4.4. Morphological Studies*

Transmission electron microscopy (TEM) analysis was carried out on a JEOL-2000 Ex II TEM (Akishima, Japan) to morphologically characterise the niosomes. The excess sample was taken off the carbon-coated copper grid using filter paper after a drop of the niosomal formulation was applied to it. The carbon-coated copper grid was then applied with a drop of 2% (*w*/*v*) PTA (phosphotungstic acid solution) and kept undisturbed for 2 min. The excess of the staining agent was removed with the help of a filter paper, and the sample was air-dried before being examined on the transmission electron microscope [63].

### *4.5. Formulation of CVC Niosomal Gel (CVCNG)*

Niosomal dispersions have a very low viscosity, which makes them unsuitable for topical use; therefore, gelling was performed. Carbopol 934 was spread out by slowly adding distilled water and letting it sit in the dark so that it could fully swell. The dispersion was further made neutral by adding triethanolamine drop by drop to achieve the intended result of a viscous transparent gel. Finally, the optimized CVC-N formulation was added slowly to the gel with constant agitation to create CVC-NG [64].

### *4.6. In Vitro Drug Release Study*

The optimized CVC-N formulation and carvacrol suspension (drug (10 mg)) was dissolved in methanol and the volume was made up to 10 mL with adequate solvent. The drug release investigation was conducted using the cellulose dialysis membrane bag (molecular weight cut off–12,000 kDa). The dialysis bag was filled with both of the prepared formulations (2 mL of each containing 2 mg of the drug), and the ends were knotted. As part of the experiment, the dialysis bag was submerged in 100 mL of the phosphate buffer (pH 6.8; medium). The release media's temperature was maintained at 37 ± 2 ◦C throughout the investigation, which was conducted with constant stirring. The sample (1 mL) was taken at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 h, and precisely the right amount of fresh medium was added to keep the sink conditions. The in vitro drug release study was conducted for 24 h to determine the drug release pattern, the mechanism it follows and how long it remains in the target area. The obtained samples were filtered (0.22 µm) and further diluted to determine the content of the drug by utilizing a UV spectrophotometer at 280 nm. To evaluate the release mechanism, the drug release data were fitted to various release kinetic models, including zero-order, first-order, Higuchi, and Korsmeyer–Peppas models [65].

### *4.7. Characterization of Gel*

### Evaluation of pH and Texture of CVC-N Gel

A digital pH meter electrode (Mettler Toledo, Chiyoda, Japan) was utilized to directly measure the pH of the CVC-N gel. TA.XT Plus Texture analyser (Stable Micro Systems Ltd., Surrey, UK) was used to determine the gel's texture. The texture analysis curve produced at the end of the trials was used to identify parameters including hardness, consistency, cohesiveness, and gel viscosity index [66].

### *4.8. Spreadability Studies*

The parallel plate method was used to determine the niosomal gel spreadability. In this procedure, 500 mg of the formulated gel was added to a circle diameter of 1 cm, atop a glass plate that had been pre-marked and another glass plate had been placed on top. Five minutes were given for a weight of 500 gm to lay on the top plate of the glass. It was seen that the gel spreading caused the diameter to expand [67].

### *4.9. Permeation Depth Study by Loading Rhodamine B Dye*

Franz diffusion cell was utilized in the confocal studies to evaluate the infiltration in the skin by rhodamine-B-dye-loaded niosomal formulation and standard solution of rhodamine B dye. A 1 cm<sup>2</sup> sample of rat skin was cut from the abdomen, and the hairs were removed using purified water and a phosphate-buffered saline solution; the rat skin was meticulously washed and rinsed until it was completely clean. The stratum corneum was then facing up and the dermis was facing down when the skin was then put on the diffusion cell. The niosome formulation was mixed with rhodamine B, while the standard was hydroalcoholic rhodamine B solution. Both were put into the donor compartment and kept at a temperature of 32 ± 0.5 ◦C for six hours. Additionally, slides with the stratum corneum pointing upward were examined using a confocal microscope and an optical excitation of 488 nm. Above 560 nm, both the argon laser beam and the fluorescence emission could be seen. Confocal laser scanning microscopy (CLSM) was used to compare the penetration and distribution of improved gel formulation with traditional gel (TCS SP5II; Leica Micro System Ltd., Wetzlar, Germany) [68].

### *4.10. Dermatokinetics*

The dermatokinetic analysis was performed to determine the quantity of drug in different layers of excised rat skin after applying the CVC-NG formulation mounted on a diffusion cell apparatus. The research was carried out following the protocol described in the CLSM study. In this case, the Franz diffusion cell skin samples were obtained for this study at 0, 1, 2, 4, and 8 h. The skin was then washed with normal saline and kept at 60 ± 0.5 ◦C for 2–3 min. The skin layers (epidermis and dermis) were separated using forceps and sliced into tiny fragments before being soaked in methanol for 24 h to extract the drug contained in the layers. The methanolic extracts of the drug were further filtered (0.22 µm), and the drug concentration was determined using a UV spectrophotometer. All the measurements were performed in triplicate [69].
