*3.5. Preparation of ITZ Self- Emulsifying Nanovesicles Gel (CMFG-ITZ- nPEVs)* 3.5.1. Extraction and Purification of Fenugreek Gum (FG)

Fenugreek seeds were cleaned, washed, and air-dried and then soaked in water overnight and the seeds were boiled in hot water for 3 h at 80 ◦C to extract the gum and inactivate the enzymes, respectively. The solution was then allowed to cool to room temperature before being pressed through a cotton towel. With the addition of an equivalent volume of acetone, the crude gum was precipitated from the ensuing viscous solution. Gum was purified by washing it in ethanol and then in acetone [33]. The purified gum was dried overnight at 50–60 ◦C in an oven. The dried gum was pulverized and sieved through mesh #100 and stored at room temperature for further use.

#### 3.5.2. Synthesis of Carboxymethyl Fenugreek Gum (CMFG)

The 1:1 ratio of FG and sodium bicarbonate mixed well in a mortar followed by the addition of ethanol (<0.01%) for the surface treatment of gel. The gel transferred to a flask fitted with a thermometer and mechanically stirred for 30 min. The solid mono-chloroacetic acid (1%) was added to the above gel in the presence of temperature 75 ◦C with continuous stirring for another 3 h. The reaction mixture was immediately cooled and neutralized to pH 7 using dilute acetic acid [34]. The gel was washed twice with methanol: water (80:20) followed by methanol washing and dried at 50–60 ◦C in oven an overnight.

#### 3.5.3. Preparation of CMFG-ITZ-nPEVs Gel

In the dark, Chen and colleagues described the synthesis of nanogel using a simple diffusion and dialysis approach [35]. In a nutshell, 50–100 mg chitosan was dissolved in 5 mL acetic acid at pH 3.0, and 50–100 mg CMFG (kept overnight for hydration in purified water) was dissolved in 5 mL dimethylformamide (DMF) by vortex and sonication (Table 2). Dropwise addition of CMFG solution into the chitosan solution was done for 12 h at 30 rpm

at room temperature, then dialyzed for 24 h with deionized water. The dialysis medium was refreshed at least five times after the free CMFG and DMF were completely removed from the solution. Finally, the solution was filtered through a 0.22 m syringe filter before being lyophilized (Christ, Alpha1-2 LD plus, Osterode am Harz, Germany) to obtain the CMFG-ITZ-nPEVs gel, which was kept at 4 ◦C for 24 h.

#### *3.6. Characterization*

#### 3.6.1. Drug Content (DC)

After disrupting the dialyzed nPEVs with methanol, the quantity of ITZ entrapped in them was determined. To create a transparent solution, an aliquot of nPEVs was combined with an appropriate proportion of methanol and then covered with a parafilm to prevent methanol evaporation [17]. After adequate dilution, the concentration of ITZ was detected spectrophotometrically (Model No. UV 2401(PC), S.220 V, Shimadzu Corporation, Kyoto, Japan) at 262.4 nm. At this wavelength, there was no interference from blank nPEVs.

#### 3.6.2. % EE

The entrapment efficiency was calculated according to the following equations-

$$\begin{aligned} \text{\%} \text{ Entrapment efficiency} &= \text{(Actual drug content in ITZ-nPEVs)} / \\ \text{\textbullet (Theoretical drug content in ITZ-nPEVs)} &\times 100 \end{aligned} \tag{1}$$

#### 3.6.3. Deformability Index

Extrusion was used to determine the deformability index of ITZ-nPEVs utilising a locally produced and approved Sartorius stainless steel pressure filter holder. The vesicles were computed using the following equation after being extruded through a membrane filter with a pore size of 50 nm at a constant pressure of 0.17 MPa [36].

$$\mathbf{D} = \mathbf{j}/\mathbf{t} \, [\mathbf{r}\mathbf{v}/\mathbf{r}\mathbf{p}]^2 \tag{2}$$

where

D is the deformability index (mL·s −1 ), j is the amount of vesicular dispersion extruded in mL, t is the time of extrusion in second, rv is the size of vesicles after extrusion (nm), and rp is the pore size of the filter (nm).

#### 3.6.4. Viscosity of nPEVs

The prepared ITZ-nPEVs and CMFG-ITZ-nPEVs gel kept overnight for hydration in milliQ water and viscosity of that preparations were determined using Brookfield viscometer (Model No. CAP2000+L, Brookfield Engineering Lab., Middleborough, MA, USA) at 100 rpm using spindle No. 1 at 37 ± 0.5 ◦C [4].

#### 3.6.5. DLS

The practical size (PS; nm), polydispersity index (PDI) and zeta potential (ZP) of ITZnPEVs and CMFG-ITZ-nPEVs gel formulations was assessed by laser Doppler anemometry in triplicate by dynamic light scattering using zeta sizer (Model No. ZS90, Malvern Instruments Ltd., Worcestershire, UK). Light scattering was monitored at 25 ◦C and 90◦ angle after appropriate dilution [37,38].

#### 3.6.6. Morphology

SEM analysis (Model No. S3700N, Hitachi, Japan) was carried out on the selected ITZ-nPEVs formula in order to characterize the shape and ultrastructure of the vesicles according to the previous method described by Moideen et al. (2020) [38].

#### 3.6.7. In Vitro Drug Release and Kinetics

The in vitro drug diffusion study of ITZ-nPEVs formulation was evaluated using USP dissolution apparatus (Type II). A dialysis membrane (average diameter; 15.9 mm, average flat width; 25.27 mm, Himedia®, Mumbai, India) was hydrated with the phosphate buffer pH 7.4 for 12 h. Volume of ITZ-nPEVs formulation equivalent to 100 mg ITZ was dispersed in 5 mL of phosphate buffer and then placed in the bag of activated membrane which was sealed from both the ends. The dialysis bag then ties to the middle of the shaft of the USP apparatus (DA-3, Veego Scientific Mevices, Mumbai, India) and paddle was put into the jar containing 300 mL dissolution medium. The temperature of the study was controlled at 37 ± 0.5 ◦C under stirring at the speed of 50 rpm. A one milliliter aliquot was withdrawn at fixed time intervals and immediately replaced with an equal volume of fresh buffer to maintain the sink condition [3]. All samples were analyzed at 261.80 nm by UV Spectrophotometry (UV 2401(PC), S.220 V, Shimadzu Corporation, Kyoto, Japan). The experiment was done in triplicate to assess the drug diffusion characteristics from nPEVs formulation. Drug release was compared with plain ITZ for which 100 mg of ITZ was suspended in the 5 mL of buffer and study was carried out similar to ITZ-nPEVs. Drug release kinetics was assumed to reflect different release mechanism of controlled release drug delivery systems. Therefore, five kinetics model were applied to analyze the in vitro data to find the best fitting equation according to our previous study.

#### 3.6.8. Nail Hydration Study

Nail clippings were collected with nail clippers from healthy human volunteers (males and females, 25–50 years old). The current investigation employed just the middle, index, and ring fingernails as an in vitro model for evaluating transungual administration [39]. Nail clippings were thoroughly cleaned by washing five times with distilled water, wiping with tissue paper, and drying at 37 ◦C for 24 h before being kept in airtight containers [40]. Fifty milligrams of nail clippings was inserted in separate glass vials for the nail hydration experiment. For this investigation, three groups were formed: group I (control group), in which the pre-weighed nail clippings were immersed in 1 mL deionized water (pH 7.04), group II (nPEVs formulation), group III (nPEVs CMFG gel) and group IV (marketed Itrostred gel), in which the nail clippings were immersed in marketed Itrostred gel, which is equivalent to the 1 mL of nPEVs. The nail clippings were reweighed after thorough tissue paper wiping to quantify weight growth after the glass vials were sealed and incubated at room temperature for 24 h [9]. The following calculation was used to compute the hydration enhancement factor (HE) after 24 h (HE24):

$$\begin{array}{l}\text{HE 24} = \text{(Weight gain of nail clipping of groups II/III/IV)}/\\\text{(Weight gain of nail clipping of group I)}\end{array}\tag{3}$$

#### 3.6.9. Transungual Drug Uptake Study

The S3 (1:7:3) was the optimized formulation of ITZ-nPEVs from the in vitro drug release and nail hydration study. Groups 2 (optimized batch S3) and 3 (Itrostred gel) nail clippings were rinsed three times with methanol to eliminate any residues of medication on the surface, then dissolved in 1 M sodium hydroxide (1 mL) with continual overnight stirring [41]. The solutions were filtered through a 0.22 m syringe filter after full digestion of the nail clippings, and an aliquot was collected and diluted with methanol before HPLC analysis. The enhancement factor (EF nail) was computed using the following equation to represent the improvement in ITZ penetration into nPEV nail clippings as compared to marketed gel (Itrostred gel)

> EF nail = (Extracted drug percentage in nail clippings of group II/III)/ (Extracted drug percentage in nail clippings of group IV) (4)

#### 3.6.10. Anti-Microbiological Efficacy

The antifungal activity of the control, a chosen batch of nPEVs formula (S3), nPEVs loaded into the gel formulation, and the commercial preparation (Itrostred gel) was tested against *Candida albicans* (MTCC No. 227). One milliliter of the fungal culture suspension was combined with 9.9 mL liquid broth (without agar) and inoculated for 24 h at 25 degrees Celsius in an incubator (Remi instruments cooling incubator, Mumbai, India). 1 mL of inoculated liquid broth containing fungal culture suspension was poured to the sterile petri dishes containing solidified agar growth medium, and the inoculum was dispersed equally across the solid agar surface by turning the plate clockwise and anticlockwise.

With the use of a sterile cork-borer, wells were formed in the centre of the plates, and each well (6 mm internal diameter) was accurately filled with either 0.1 mL of control, ITZ-nPEVs, CMGF-ITZ-nPEVs gel, or Itrostred gel corresponding to ITZ dosage in the 0.1 mL ITZ-nPEVs. The plates were then incubated in the incubator for three days at 25 ◦C to allow for fungal development [42]. The antifungal activity was determined by measuring fungal growth inhibition zones around the formulations. They were measured on mm scale, and a comprehensive antifungal analysis was performed in an aseptic environment.

#### 3.6.11. Stability Study

The physical and chemical stability study of an ideal formulation of CMFG-ITZ-nPEVs at 45 ± 0.5 ◦C and 60 ± 5% RH for 3 months in stability chamber (Model No. HTC-3003, Wadegati TM Labe Quip (P) Ltd., Andheri (E), Mumbai, India). At an interval of 1 month, CMFG-ITZ-nPEVs gels analyzed for physical changes, drug content, particle size, zeta potential and in-vitro drug release.

#### **4. Conclusions**

The combination of the penetration enhancer labrasol, the nail penetration enhancer N-acetyl-L-cysteine, and the positive charge inducer stearylamine in aqueous deformablenatured nanovesicles (nPEVs) was proven to be a viable combination for improving ITZ transungual administration. The prepared CMGF-ITZ-nPEVs gel satisfy best attributes for topical application such as it spreads easily, exhibiting maximum slip and drag. This nanosized formulations enhance permeability of drug extend retention at the site of action and the CMGF-ITZ-nPEVs gel shown greater antifungal activity than the marketed gel. The ITZ-nPEVs revealed here provide a novel therapy option for nail-related illnesses such onychomycosis. Clinical trials to determine the effectiveness of ITZ-nPEVs in patients with onychomycosis are presently underway.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/polym14020325/s1, Figure S1: (**A**) Quantification chromatogram of ITZ, and (**B**) standard calibration curve of ITZ; Figure S2. (**a**) PS and PDI, (**b**) ZP of S3; Table S1: Peak area of ITZ at different concentration.

**Author Contributions:** Conceptualization, Investigation, Methodology, Writing—original draft made by B.R., S.K. and J.M.M.M. Funding acquisition, Software, Resources and Visualization done by A.A. (Ali Alqahtani), A.A.F., T.A. and A.A. (Ali Alamri). Formal analysis, Data curation, writing—review and editing, statistical analysis and validation carried out by F.A. and V.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful to the King Khalid University's Deanship of Scientific Research for sponsoring this study through the Large Research Group Project, under grant number of RGP2/186/42.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data sharing not applicable.

**Acknowledgments:** The authors are grateful to the King Khalid University's Deanship of Scientific Research for sponsoring this study through the Large Research Group Project, under grant number of RGP2/186/42.

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

## **References**

