Materials

KTZ was obtained as a gift sample from Modex Pharma Ltd. (Gujarat, India). Beeswax, Carnauba wax, and lanolin anhydrous (LR) were purchased from S.D Fine Chemicals Ltd. (Maharasthra, India). Tween 80, PEG-400, butylated hydroxyanisole (BHA), and transcutol P were purchased from Finar Ltd. (Gujarat, India). Titanium dioxide was purchased from Qualigens Fine Chemicals Pvt. Ltd. (Maharasthra, India). Iso-propylmyristate (IPM) and methanol were obtained from Chemdyes Corporation (Gujarat, India) and Central Drug House Ltd. (New Delhi, India). Cow ghee and cold press castor oil were obtained from the Ashtamangal Oil mill (Maharasthra, India). These materials were food grade, and all the other chemical reagents were of analytical grade.

Screening of Excipients for the development of SEDDs

The selection of excipients was done based on the solubility of KTZ in the various oils, surfactants, co-surfactants, and co-solvents. The solubility study was carried out by incorporating the excess quantity of KTZ in different excipients and mixing vigorously with the help of a magnetic stirrer. These mixtures are then stored at room temperature for 24 h for equilibration, followed by centrifugation at 5,500 to 6,000 rpm for 15 min. The supernatant was filtered, diluted, and analyzed using a UV-1800 UV–visible spectrophotometer (Shimadzu, Kyoto, Japan) at 220 nm. λ max of KTZ for determining the solubility [24].

Construction of pseudo-ternary phase diagram

The pseudo-ternary phase diagram was plotted for finding out the formulation which is suitable for generating the oil-in-water emulsions. Based on solubility studies, IPM, Tween 80, and PEG 400 were chosen as oil phase, surfactant, and co-surfactant. The phase diagram was constructed by employing the water titration method wherein the Smix (Tween 80: PEG 400) was taken in 1:0.5, 1:1, 1:1.5 ratios. The different Smix ratios were mixed with oil in specific combinations of 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, and 1:9 using a magnetic stirrer set to 900 RPM and 40 °C. This is followed by dilution of 0.2 mL of formulation up to 100 times with distilled water under continuous stirring. The phase diagrams were plotted using Sigma Plot 12.0 (Systat Software Inc., USA) [25, 26].

Preparation of KTZ-SEDDs

KTZ-SEDDs were developed using a one-step emulsification method (Fig. 1). In this method, KTZ (0.2 g) was mixed with IPM (1.85 g) and heated at 40 °C with continuous stirring at 900 rpm for preparing the oil phase. To the above phase, Tween 80 and PEG 400 were added followed by heating the mixtures at 40 °C and stirring until a clear homogeneous solution is produced. The SEDDs were allowed to cool down to room temperature and emulsification was tested by diluting 0.1 g of the sample up to 200 times with deionized water. Oil-in-water emulsion could be spontaneously generated in the aqueous solution which confirms the self-emulsification tendency of the optimized combination [25, 26].

Fig. 1

Preparation of the KTZ-SEDDs and transformation of SEDDs into lipstick

Development of lipstick from KTZ-SEDDs using experimental design

The combined effect of three independent variables, i.e., amount of beeswax (X1), carnauba wax (X2), and cow ghee (X3), was evaluated using a Box–Behnken design. The concentrations of the variables were taken in three levels, i.e., upper (+ 1), middle (0), and lower (-1) limits (Table 1). The randomized run presented 15 different formulation batches (F1 to F15) is represented in Table 2, wherein F7, F10, and F13 are triplicates of central values. The quantity of independent variables given in Table 1 is selected based on a literature review and preliminary trials. The dependent variables or responses chosen for the experiment were softening point (Y1) and percentage drug release after 12 h (Y2). The models were validated by lack of fit, ANOVA, and multiple correlation coefficient tests. The experimental design was conducted using Design-Expert® 13 (version 13.0.5.0, Stat-Ease Inc., Minneapolis, USA).

Table 1 Variables in the experimental designTable 2 Composition of formulations as per randomized runs in Box–Behnken design

The prepared SEDDs were transformed into lipstick formulations for topical delivery of KTZ. For this purpose, the weighed quantities of lanolin, beeswax, and carnauba wax were melted in a porcelain dish in the water bath to prepare the melted wax mixture (mixture 1). Similarly, the cow ghee was melted in a separate porcelain dish, and castor oil was added to it (mixture 2). A measured quantity of SEDDs was incorporated into mixture 1, followed by the addition of mixture 2 along with vigorous stirring until a soft emulsion is generated. The quantity of components used in the preparation of 15 formulations is given in Table 2. The molding of the emulsion into lipstick was done by adding it to previously lubricated and chilled molds. The solidified lipsticks were removed from the mold by using a scrapper and reshaped from the edges (Fig. 1).

Characterization of KTZ-SEDDs lipstick formulationMelting point

The lipstick sample (about 2 g) was taken in a glass capillary tube. The tube was immersed in the beaker-filled water, which was placed over the water bath for heating. The melting point was estimated as the temperature at which the material starts to melt and forms a liquid drop [27].

Breaking load test

The strength of the lipstick formulation was assessed by the breaking load test. In this, the lipsticks were held horizontally into an opening close to the edge of the support, and the weights attached to it were gradually increased (10 g) at a specific interval of 30 s. The weight at which the lipstick breaks is recorded as the breaking point weight [28].

Spreadability test

The spreadability of lipstick was assessed by spreading it over a transparent glass slide at an angle of 45°. The surface of the slide was observed visually against the dark background for its uniformity in forming a consistent layer, fragmentation, or deformation upon application. The following criteria laid down for this test are the symbol ‘G’ indicates a Good and uniform layer without fragments and deformation, the symbol ‘M’ indicates moderate uniformity with few fragments, but no deformation, and ‘P’ indicates poor uniformity with high fragmentation and clear deformation [29,30,31].

Surface anomalies

The quality of lipstick may be indicated by estimating the surface anomalies. The samples were examined visually to check for anomalies that may occur on the surface of the lipstick. In this study, the lipsticks were checked for marks (representing scratches or dents on the surface), heterogeneity (color or texture differences), pollutants (dust or other undesirable particles), and distortion (irregular line, deformation, etc.). The surface of the product should be completely devoid of crystal formation with no signs of contamination from the molds and fungi [32, 33].

Percentage drug content

For the determination of drug content, a weighed quantity of lipstick (100 mg) sample was transferred into a 100-mL volumetric flask and dissolved into appropriate amount of methanol. The absorbance was measured using a UV–Vis spectrophotometer at 220 nm after suitable dilution [34, 35].

Softening point

The softening point test was performed by inserting the lipstick sample into an aluminum ring, and extra mass above and below was removed. The ring containing the sample was placed in the refrigerator at 6 °C for 10 min. After removal, the ring was fastened into a stand and dipped into a beaker filled with water. The whole assembly was heated with continuous stirring and the softening point was recorded as the temperature at which the lipstick mass loosened and fell into the beaker [36, 37].

Permeability study

The lipstick formulations were tested for permeability using vertical glass Franz diffusion cells (Electrolab Private Limited, India). The transparent jacketed cell with a flat bottom and a 5-mL receptor was used for the experiment. The release of KTZ was studied by employing a cellophane membrane (Sigma-Aldrich, 0.22 µm pore size). The mixture of deionized water and methanol was selected as receptor media which was filled in the receptor compartment and magnetically stirred at 600 rpm. The experiment was conducted at 32 ± 0.5 °C for 12 h involving withdrawing samples at 1-h interval and analyzed using a UV spectrophotometer at 220 nm [38, 39].

Stability study

The stability of lipstick was estimated at room temperature (25 °C), 40 °C, and 45 °C in the stability chamber for 4 weeks. Samples of the selected formulation were examined for their melting point, spreadability, breaking point, surface anomalies, drug content, and percentage drug release after 12 h after completion of the 1st, 2nd, and 4th week [40].

In vitro antifungal activity

The antifungal activity of the formulation was studied using the cup-plate method. The dextrose agar media was dissolved in 100 mL of distilled water with the help of continuous stirring and heating. The solution was boiled for some time for effective dissolution, followed by maintaining pH at 5.5 and autoclaving at a temperature of 121 °C for 15 min. The media was poured into two sterile Petri plates in an aseptic area and the plates were set aside for solidification. After solidification, the Candida albicans were inoculated on the surface of media in both plates and small holes were created. In one of the plates, 1% w/v KTZ solution in ethanol was placed in the holes as a reference. The formulation was subjected to the holes of agar media in another plate and both plates were incubated for 24 h at a temperature of 25 °C. The plates were removed after 24 h, and the zone of inhibition was estimated in millimeters by utilizing a vernier caliper. The diameter of the zone of inhibition was recorded, and each measurement was performed in triplicate [41, 42].

Statistical analysis

Statistical analysis was performed using Design-Expert® 13, and one-way ANOVA was performed for the statistical comparisons. The analysis was done using statistical parameters such as multiple correlation coefficient (r2), adjusted multiple correlation coefficient (adjusted r2), coefficient of variation (C.V.), and lack of fit proven by Design-Expert software.