Materials

Miconazole nitrate (MZN) was kindly supplied by Medical Union Pharmaceuticals, Egypt. Brij 35, Brij 58, Brij 97, and dialysis cellulose membrane (cut-off 14,000 g/mol) were purchased from Sigma-Aldrich, USA. Acros Organics, USA, supplied Span 60, Tween 20, Tween 60, and Tween 80. Propylene glycol 4000 was obtained from Loba-Chemie, India. Triethanolamine and ethanol were purchased from ADWIC, Egypt. Carbopol 934 was acquired from Goodrich Chemical Company, USA. The buffer constituents were obtained from El-Nasr Pharmaceutical Chemicals Co., Egypt: disodium hydrogen phosphate and potassium dihydrogen phosphate. All other solvents and chemicals were of analytical grade and were purchased and used as received.

MethodsPreparation of Miconazole Nitrate-Loaded Spanlastics Using D-Optimal Design

The composition of the various MZN-loaded spanlastics is listed in Table 1. The materials used in preparing the spanlastics are considered safe (GRAS), besides being FDA-approved [31]. Span 60, a non-ionic surfactant (SAA), was selected as the membrane-forming material. In addition, a range of non-ionic SAAs with various HLB values were employed as edge activators (EAs). Generally, the non-ionic surfactant class was selected owing to its safety, compatibility, and stability relative to the other surfactant classes [31].

Table 1 Composition of the various formulas of MZN-loaded spanlastics and the responses measured. Data express the mean ± range, where n = 3

Due to its simplicity and reproducibility, the ethanol injection method was adopted to prepare MZN-spanlastics [32, 33]. MZN (0.2 g) and the surfactant Span 60 were dissolved in 3 mL of 100% ethanol. The ethanolic solution was gently injected into a warmed aqueous medium containing the EA and constantly stirred on a magnetic stirrer (Jenway 1000, Jenway, UK) for 30 min until spanlastics dispersion (10 mL) was formed. The temperature was set to 80 °C and the stirring speed to 1000 rpm. The total SAA percentage in the formula (Span 60 and EA) was kept constant at 2% (w/v) of the total formulation volume (10 mL). In addition, the Span 60 percentage was varied in relevance to the EA percentage so that their sum would make 100% of the total SAA percentage used in the formula. To ensure that all of the alcohol was removed, the dispersion was stirred for another hour at the same room temperature and speed. The prepared formulas were left to mature overnight and then stored at 4 °C for further studies [34].

Experimental Design

MZN-loaded spanlastics were prepared as proposed by D-optimal design using Design-Expert® 12.0.3.0 software (Stat-Ease Inc., USA) to examine the influence of formulation variables on the vesicle properties. In this design, two independent factors were evaluated, namely the percentage of edge activator (EA) (A) and the type of EA (B). The dependent variables were particle size (PS) (R1) and % EE (R2). The first independent factor was studied at 2 levels (10 and 30%) and 1 center point (20%), whereas the second was studied at 6 levels, namely Brij 35, Brij 58, Brij 97, Tween 20, Tween 60, and Tween 80, where 17 possible combinations were experimented as shown in Table 1 [35]. A summary of the factors and their levels is presented in the supplementary material, Table S1.

Model Selection and Validation

The best-fitted models for both R1 and R2 were selected based on the lowest model p-value (significant), highest R2 value, highest lack of fit p-value (non-significant), and where the difference between the predicted R2 and the adjusted R2 is in reasonable agreement (less than 0.2) [36, 37].

Optimized Formula Selection and Evaluation

Based on the desirability approach, numerical optimization was carried out by selecting PS between 150 and 250 nm and maximizing the % EE. All factor weights were equal and set to ½. [38] The optimized formula, proposed by the software was prepared in triplicate and assessed by re-measuring the dependent variables and comparing them with the expected results, yielding the percentage bias; consequently, the validity of the design was checked. The optimized formula was subjected to further investigations such as drug released percentage, physico-pharmaceutical characterization, and an ex vivo skin deposition test. For some tests, an adequate volume of the prepared aqueous dispersion was lyophilized to obtain it in powder form. The F7 aqueous dispersion was transferred to a 15-mL falcon tube and frozen overnight at −20 °C. After that, the sample was lyophilized using a Novalyphe-NL 500 freeze-dryer (Savant Instruments Corp., USA) for 24 h under a pressure of 7 × 10−2 mbar at −45 °C. The fluffy powder obtained was then kept in a dry, cool place for further use.

Statistical Analysis

Statistical analysis was performed using Design-Expert® 12.0.3.0 software. Data was analyzed using one-way analysis of variance (ANOVA), and differences were considered significant when p was less than 0.05.

Characterization of Miconazole Nitrate-Loaded Spanlastics Particle Size, Polydispersity Index, and Zeta Potential Measurements

Dynamic light scattering was utilized to determine the average particle size (PS) and polydispersity index (PDI) of the developed particles at 25 °C using a Malvern Zetasizer (Nano-ZS, Malvern Instruments, Malvern, UK). Before measuring the PS, all formulations were adequately diluted with distilled water to guarantee a proper scattering intensity [39, 40]. The PDI was employed to indicate the homogeneity of the formulas, where a small value implies a homogenously sized vesicle and vice versa. The measurement was conducted three times for each sample, and the mean ± SD was calculated.

The vesicles’ zeta potential (ZP) was assessed using the same instrument by detecting their electrophoretic mobility in an applied electrical field. The ZP measurements were determined in double-distilled water. Three replicates for each formula were taken [39, 41]. The ZP indicates the physical stability of the prepared particles, where a value above +30 mV or below −30 mV suggests the stability of the particles.

Entrapment Efficiency

To determine the amount of MZN entrapped in the formulas, the MZN-loaded vesicles were centrifuged at a rotation speed of 15,000 rpm and a temperature of 4 °C for 1 h (Laborzentrifugen, 2k15, Sigma, Germany). The supernatant was then separated from the particles, and the amount of the free drug was measured spectrophotometrically at λmax 271 nm (Shimadzu UV spectrophotometer, 2401/PC, Japan). The entrapped percentage (EE%) of the drug was then calculated from Eq. 1 [41]:

$$\textrm\ \left(\%\right)=\frac}-_}}}}\times 100$$

(1)

where Dt is the total amount of drug, and Du is the amount of entrapped drug [42, 43].

Elasticity Measurement

The extrusion technique Van den Bergh et al. [44] described was followed to assess the elasticity of the optimum formula for spanlastics aqueous dispersion (F7). F7 was diluted and extruded through a 220-nm pore-size microporous filter (Jinteng Experiment Equipment Co., Ltd., China) under a constant pressure of 2.5 bar (Haug Kompressoren AG; Büchi Labortechnik AG, Flawil, Switzerland). The particle size was compared before and after extrusion, and the percentage of deformation was calculated [45].

Physico-pharmaceutical Characterization of the Optimized Formula Transmission Electron Microscopy (TEM)

The morphological examination of the optimized MZN spanlastics formula was carried out using a transmission electron microscope (JEM-2100, Jeol, Japan) at an acceleration voltage of 80 kV. The vesicles were properly diluted, and one drop was applied to a carbon-coated copper grid surface and dehydrated. Phosphotungestic acid, at a concentration of 2%, was used to stain the sample, which was dehydrated again at ambient temperature to give a good contrast upon TEM inspection [41].

Differential Scanning Calorimetry (DSC)

The thermograms of pure MZN powder and lyophilized F7 (the optimized MZN-spanlastics formula) were studied using a differential scanning calorimeter (DSC-50, Kyoto, Japan). An amount of 2 mg of each sample was loaded in aluminum pots, and the temperature was raised gradually at a rate of 10 °C/min from 25 to 300 °C under an inert nitrogen flow of 25 mL/min [41, 46].

Incorporation of MZN and Optimized Formula F7 in Carbopol Gel Preparation of Carbopol Gel

A Carbopol gel was formulated to incorporate the F7 and free medication within it. A quantity of 2 g of Carbopol 934 was dispersed in distilled water using a magnetic stirrer operating at 800 rpm for 60 min. The addition of propylene glycol 4000 (20 g) was carried out incrementally to the mixture. The addition of triethanolamine was carried out in a dropwise manner to achieve neutralization of the mixture. Manual stirring was performed using a glass rod until gelation occurred, and the resulting solution’s pH was measured [47].

Incorporation of Free MZN and Optimized MZN-Spanlastics in Gel

The prepared gel was used as a vehicle for the free drug and optimized formula, where an amount of 0.6 g of the drug was added to 15 g of the gel, and the weight was completed to 30 g with distilled water. Therefore, the MZN percentage in the gel was 2%.

Similarly, the optimized MZN-spanlastics in an amount equivalent to 0.6 g of the free drug was incorporated into the gel, and likewise, the MZN percentage in the spanlastics gel was 2% [41].

Characterization of Gel Formulas Visual Inspection

The prepared gel formulations’ color, homogeneity, consistency, and spreadability were visually inspected [47, 48].

pH

To determine the pH of the formula, a sample of the produced gel weighing 1 g was weighed and completed to 10 g with distilled water. The pH of the diluted sample was measured using a Jenway pH-Meter (model 3510, UK) [47, 48].

Viscosity

The viscosity of the prepared gels was measured using a Brookfield rheometer with a spindle CP 41. The viscosity test was performed at room temperature with a rotating speed of 1 rpm [47, 48].

Drug Content

An amount of 100 mg of the gel formulations was dissolved in 20 mL of ethanol and filtered, and the volume was completed with 10 mL of distilled water in a volumetric flask. The drug concentration in the solution was detected spectrophotometrically at 271 nm [47].

In Vitro Drug Release Studies of the Optimized Formulas in Aqueous Dispersion and Gel Form

The in vitro drug release profiles were performed using the membrane diffusion method for the following samples: the optimized MZN-spanlastics aqueous dispersion formula (F7), MZN-spanlastics gel (F7 gel), MZN gel, and MZN aqueous suspension. Before commencing the experiment, the cellulose membrane was soaked in a phosphate-buffered saline solution (pH = 5.4) for 24 h at room temperature. Afterward, 1 mL of each sample (or an amount equivalent to 20 mg of the drug) was loaded into the cellulose membrane, and both ends of the membrane were tightly tied. The loaded membrane was dropped into a beaker containing 300 mL of a phosphate-buffered saline solution with a pH of 5.4. The beaker was covered with parafilm and placed in an incubator shaker (IKA KS 4000, Germany). The shaker operation speed was 50 rpm, and the temperature was 37 ± 0.5 °C. At specific time intervals, aliquots of 5 mL were withdrawn and substituted by an equivalent volume of fresh buffer solution to maintain a constant volume. The withdrawn samples were analyzed spectrophotometrically at λmax of 271 nm to determine the percentage of the released drug. The results are presented as mean values ± SD of the percentage of drug release as a function of time for a total of three repeats per experiment [34]. The release data were fitted to various release kinetic models: zero, first, Higuchi diffusion, Hixson-Crowell, and Korsmeyer-Peppas models.

In Vitro Antifungal Activity Study of the Optimized Formulas in Aqueous Dispersion and Gel Form In Vitro Antifungal Activity by Well Diffusion Assay

The in vitro antifungal assay was used to determine and compare the samples’ antifungal properties against C. albicans (ATCC® 10231). The tested samples were MZN suspension, MZN gel, the optimized MZN-loaded spanlastics (F7) aqueous dispersion, and F7 incorporated in the gel. The yeasts from a 24-h culture on Sabouraud dextrose agar (SDA) were subcultured to create the inoculum. The culture was left in the incubator overnight; afterwards, 5–6 colonies of the organism were transferred to 0.9% saline, and the suspension turbidity was adjusted to match the 0.5 McFarland standard (9.95 mL of 1% sulfuric acid (H2SO4)) according to the Clinical and Laboratory Standards Institute (CLSI) protocol. A fresh SDA medium was prepared according to the manufacturer’s recommendation and poured into plates at a depth of 5 to 6 mm, and the plates were left to air dry for 30 min. The adjusted culture in saline was swabbed on the prepared SDA plates in three different directions to ensure complete coverage of the agar surface. The antifungal assay was done using both well and surface agar assays. As for the well agar assay, four wells, each 4 mm in diameter, were cut out of the agar using a sterile cork borer, and 100 μL of the tested sample (20 mg/mL) was placed into each well.

On the other hand, in the surface agar assay, 10 μL of the tested antifungal agent was placed onto the agar surface and left to dry. Plates were incubated at 35 °C for 24 h. Zones of complete inhibition were measured in millimeters. The assay was repeated three times for confirmation [49, 50].

In Vitro Antifungal Activity by Broth Microdilution Method

The minimum inhibitory concentration (MIC) was determined using the broth microdilution method for MZN-containing samples against C. albicans (ATCC® 10231), namely MZN suspension, MZN gel, F7 aqueous dispersion, and F7 gel [51]. The media utilized in this test was prepared, similar to the “In Vitro Antifungal Activity by Well Diffusion Assay” section; the cultures were incubated overnight, and their turbidity was adjusted with 9.95 mL of 1% sulfuric acid (H2SO4) to match a 0.5 McFarland standard. Finally, the media were further diluted to give a final concentration equivalent to 106 CFU/mL according to the CLSI protocol. On the other hand, the stock solution of MZN-containing samples (20 mg/mL) was serially diluted using sterile Sabouraud dextrose broth (SDB) to concentrations between 0.48 and 500 μg/mL.

One hundred microliters of the adjusted bacterial culture were added to an equal volume of the diluted MZN samples in a 96-well microtiter plate. Two controls were utilized: a negative control (Sabouraud broth only) and a positive control (Sabouraud broth with C. albicans) to confirm medium sterility and organism viability, respectively. The plates were then incubated at 35 °C for 24 to 48 h. The effect of loading MZN into spanlastics and their gel formulations was determined by measuring the change in the MIC of pure MZN. This loaded formulation’s synergistic or antagonistic activity was reflected by a decrease or increase in the MIC values, respectively, compared to MZN suspension.

The MIC was recorded as the highest dilution of the antifungal agent, which gave no visible growth. Breakpoints for C. albicans were set by CLSI for azoles as follows: itraconazole (susceptible, MIC < 1 μg/mL and resistant, MIC ≥ 1 μg/mL) and fluconazole (sensitive, MIC ≤ 2 μg/mL and resistant, MIC ≥ 8 μg/mL). There was no defined breakpoint for miconazole; however, previous research indicated that Candida sp. is susceptible and resistant at MIC ≤ 5 μg/mL and MIC > 5 μg/mL, respectively [52, 53]. The experiment was conducted in triplicate. The mean and the standard deviation were recorded.

In Vitro Cytotoxicity Studies of Optimized Formulas in Aqueous Dispersion and Gel Form

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] test was used to assess cell viability in human skin fibroblast (HSF) cell lines. First, HSF cells (passage 10) were seeded in 96-well plates at a density of 5000 cells/well in full Dulbecco’s modified eagle (DMEM) media with 10% fetal bovine serum (FBS) and 100 g/mL penicillin and streptomycin for 24 h at 37 °C in a 5% CO2 incubator. The cells were then treated with a range of concentrations of MZN suspension, F7 aqueous dispersion, and F7 gel for 24 h at 37 °C and 5% CO2. A volume of 100 μL of MTT/DMEM mixture with a ratio of 1:9 was applied to each well and incubated at 37 °C for 4 h to allow metabolically active cells to generate formazan crystals. After removing the medium, 100 μL of solubilized agent (DMSO) was added, and the optical density was measured spectrophotometrically at a wavelength of 570 nm (OD570). The viable cell percentage is directly proportional to the OD570 [54].

Ex Vivo Study of Gel Containing MZN and MZN-Spanlastics: Skin Deposition Test

Male Sprague Dawley rats weighing 220–240 g were sacrificed, and the back skin was meticulously removed using electrical clippers in accordance with ethical guidelines. The protocols of the conducted study were all approved by the MSA research and ethics committee (application number PT15/Ec15/2020F). Using a scalpel, the subcutaneous fats and cartilage were separated from the skin, which was then cleaned with phosphate-buffered saline, wrapped in aluminum foil, and frozen until needed.

Before use, the rat skin was given an hour to soak in phosphate buffer (pH 5.5) and divided into the proper sizes. The skin deposition experiment was performed on a Franz diffusion cell apparatus (MicroettePlus™; Hanson Research, Chatsworth, USA). The stratum corneum side of the skin was mounted in open, two-chamber Franz-type diffusion cells that were filled with phosphate buffer saline, pH 5.5. A thermostatically controlled water bath was used to place the diffusion cells, resulting in a skin surface temperature of 37 °C.

A Teflon-coated magnetic bar was used for stirring in the receptor compartment, which was filled with 10 mL of buffer; the stirring speed was set to 200 rpm. The stratum corneum surface was then covered with 1 g of the optimized formula and plain MZN gel. Experiments were conducted in triplicates, and the results from each experiment were presented as an average.

Following the incubation period, skin samples were taken out and thoroughly cleaned thrice using phosphate buffer soaked to remove any formulations that might have remained. The skin was then separated into three layers: stratum corneum, dermis, and epidermis. The stratum corneum was separated using a tape-stripping technique, whereas a pair of forceps was used to separate the dermis from the epidermis. Absolute ethanol was used to extract any drug retained on each skin layer sonicated for 30 min. Then, the skin extracts were filtered using 0.47 μm Millipore filters and injected into the HPLC coupled with an ultraviolet detector (Model SPD-10 A; Shimadzu) to quantify the drug content at λmax of 220 nm. The used column was Inertsil ODS: 4.6 × 250mm, 3.5 μm, and a mixture of water and methanol with a ratio of 15:85 was utilized as the mobile phase. The sample flow rate and temperature were set to 0.8 mL/min and 25 °C, respectively [41, 55, 56].

Stability Studies of Spanlastics Aqueous Dispersion

The change of the particle size as a function of time determines the formula’s stability. Thus, the optimized formula was kept in the refrigerator for 6 months, and the particle size and drug entrapment efficiency were re-measured and compared to the freshly prepared samples [57].