The materials used in this study were vemurafenib (Hangzhou Hyper Chemicals, China), peppermint oil (BAR_SUR_LOUP, France), PEG 400 (Vardaan House, India), 1-tetradecyl-3-methylimidazolium bromide (C14MIB) and HPMC K15M (Hyperchem, China), potassium dihydrogen phosphate, disodium hydrogen phosphate and hexadecyltriammonium bromide (HTAB; Hejazifar et al. 2020; Himedia, India).

Phase behaviour study was conducted to estimate the ME area and predict the quantities of each SAIL (C14MIB), co-surfactant (PEG 400) and oil (peppermint oil) used to prepare a stable ME. The procedure included using different SAIL: co-surfactant (Smix) ratios, including 1:2, 1:3 and 1:4 w/w. Each of the previously mentioned Smix ratios was mixed separately with peppermint oil in different weight ratios (i.e. 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 w/w). Water was added to each oil: Smix ratio with gentle stirring until turbidity appeared. The amount of water (in gm) added in each oil: Smix ratio was recorded, and the weight percent of each oil: Smix: water was recorded and used to prepare the pseudoternary phase diagram (Maraie and Almajidi 2018; Farooq et al. 2019).

MEs were prepared by dissolving 10 mg of vemurafenib in a different mixture of peppermint oil and Smix and stirred until the drug was completely dissolved in the oil: Smix mixture. Subsequently, the required amount of water was added dropwise for 10 min until a homogenized translucent mixture was achieved. The prepared formulas were left for 24 h to equilibrate (Naeem 2019). Nine formulas (CP1–CP9) were prepared using Smix ratios of 1:4, 1:3 and 1:2. Formulas CP1, CP2 and CP3 were prepared using a Smix ratio of 1:4. Formulas CP4, CP5 and CP6 were prepared using a Smix ratio of 1:3. Formulas CP7, CP8 and CP9 were prepared using a Smix ratio of 1:2. Formulas CP3, CP6 and CP9 were prepared using 15% w/w peppermint oil, whereas other formulas contained 10% w/w peppermint oil. Formulas CP2, CP5 and CP8 contained 70% w/w Smix, and other formulas contained 60% w/w Smix (Table 1).

The Zetasizer apparatus (Malvern, the UK) was used to measure the particle size and PDI for all formulas (CP1–CP9). About 1 mL of each sample was obtained and diluted up to 3 mL with deionized distilled water (to ensure that sample viscosity was comparable with water viscosity and facilitate free movement of ME particles). The diluted formulas were placed in the instrument, and the particle size and PDI were recorded for each formula (Basheer et al. 2013; Sukre et al. 2022). Zeta potential was measured using the same instrument, and only formulas that passed all tests were used to prepare ME-based hydrogels.

MEs were examined visually to confirm their translucency and the absence of any turbidity or coalescence. Thereafter, the prepared MEs were subjected to centrifugation at 3000 rpm for 5 min and examined for any separation or turbidity to confirm that the prepared MEs could withstand vigorous shear stresses.

Dilution tests were performed to predict whether the prepared ME was o/w or w/o and could be diluted in a gel base. This test was performed by adding deionised distilled water to 1 mL of each prepared formula, and their clarity and the absence of any turbidity were observed (Nirmala et al. 2013).

EC was measured using a conductivity meter (TDS Ec Meter Temperature Tester, China) by placing the device electrode in the prepared SAIL-based MEs. The electrical current was recorded and measured in µS/cm (Changmai et al. 2019).

The physical stability of the MEs was determined through two tests. The first test was called the heating–cooling cycle. In this test, the prepared SAIL-based ME was subjected to different temperatures (4 °C, 25 °C and 40 °C) for at least 48 h for each temperature. The second test called the freeze–thaw cycle included storage of the prepared SAIL-based ME at -20 °C and 25 °C for 48 h at each temperature. This cycle was repeated three times. Thereafter, the MEs were inspected for any possible turbidity or separation (Bergonzi et al. 2014; Ghareeb 2020).

pH was measured using a pH meter device (HANNA RI 02895, Romania). The instrument was calibrated at pH 7 and 4 using standard buffer solutions. The probe of the device was dipped in each prepared ME (CP1–CP9), and the resultant pH was recorded (Sukre et al. 2022).

The drug content of each prepared SAIL-based ME (100 mg) was determined by diluting it with methanol to a concentration that could be detected spectrophotometrically (10 mL) and measuring the concentration at λ_max 305 nm (Yadav et al. 2017). The drug content of each formula was measured using the actual and theoretical drug contents via the following equation:

$\text{Drug content} (\%)=\frac{ Actual drug content}{Theoretical drug content}*100\%$ (1)

In vitro drug release was determined using the dialysis bag method. The dialysis bag had a pore size between 8,000 and 12,000 Da. The dissolution medium was the same FDA dissolution medium of vemurafenib that consisted of 1% HTAB (Hejazifar et al. 2020) in 0.05 M phosphate buffer, pH 6.8. The procedure included hydration of the dialysis bag with the previously mentioned dissolution medium for 24 h. Thereafter, 1 g of each formula was placed in the hydrated dialysis bag and sealed from each side. The loaded dialysis bag was placed in 100 mL of dissolution medium and rotated at 50 rpm at 37 °C. About 3 mL of sample was withdrawn from the dissolution medium after 1, 2, 4, 6, 12 and 24 h. Vemurafenib absorbance was measured at λ_max 307 nm, and the concentration of vemurafenib in each sample was estimated using a calibration curve equation. The percent of the cumulative amount released was predicted from the estimated concentration (Huang et al. 2008; Dawood et al. 2018).

The ME-based hydrogels were prepared using HPMC K15M as the gelling agent. The required amount of HPMC K15M was gradually added to boiled deionised water while stirring at 15000 rpm. The concentration of HPMC K15M prepared was 5% and 7% w/v HPMC K15M. The selected formula of the cold MEs (CP5, CP7 and CP8) was added to the prepared boiled HPMC K15M mixture in a ratio of 1:1 w/w with continuous stirring. The prepared ME-based hydrogels were stored in the refrigerator for 24 h for complete relaxation and gel formation (El-Say et al. 2016). Six formulas (GP1–GP6) of ME-based hydrogels were prepared. The ME used in GP1 and GP2 was from CP5. The ME used in GP3 and GP4 was from CP7, and the ME used in GP5 and GP6 was from CP8 (Table 2).

ME-based hydrogels (GP1–GP6) were inspected for their transparency (or translucency), homogeneity, general consistency and phase separation (Almajidi et al. 2022).

pH was measured using a pH meter device (HANNA RI 02895, Romania). The instrument was calibrated at pH 7 and 4 using standard buffer solutions. The probe of the device was dipped in each prepared ME-based hydrogel (GP1–GP6), and the resultant pH was recorded (Jaber et al. 2020).

The parallel plate method was used to estimate the spreadability of ME-based hydrogels. About 1 g of each prepared ME-based hydrogel (GP1–GP6) was placed on the centre of a glass plate (20×20 cm) that was placed on white paper. Another glass plate of the same size was placed on the first plate, and a 2 kg weight was placed on the second plate. The sample was allowed to spread between the two plates for a few minutes until no further spreading was expected (Jaber et al. 2020). The spreadability capacity (g·cm/s) was measured using the following equation:

$Spreadability \left(\mathrm{g}·\frac{\mathrm{cm}}{\sec }\right)=\frac{ weight placed \left(\mathrm{gm}\right)\times \text{ length of sample migration }\left(\mathrm{cm}\right)}{ Time \left(\sec \right)}$ (2)

A viscosity and rheology study was conducted by using a Brookfield digital viscometer (NDJ–5S). The spindle (spindle No. 4) of the device was placed in each ME-based hydrogel prepared (GP1–GP6) and rotated at different speed rates (6, 12, 30 and 60 rpm) for 30 s at each speed. The viscosity was recorded at each speed (Sabri et al. 2009).

The skin permeability study was performed using the abdominal skin of Wister Albino rats with the University of Baghdad–College of Pharmacy ethics committee. The experiment was performed using Copley^® Franz cell (Nottingham, the UK). The receptor cell was filled with FDA dissolution media of vemurafenib (1% HTAB in 0.05 M phosphate buffer, pH 6.8), and skin was placed on the top of the receptor cell. The skin’s outer layer was facing the donor compartment, and the inner part was facing the receptor part of the cell. A ring (whose cavity had a surface area of 1.76 cm²) was placed on the skin’s outer layer. About 200 mg of hydrogel was placed in the ring orifice and spread uniformly. The system was sealed by placing a cover and sprig above the ring to prevent any contamination of the sample. The prepared Franz cell was placed in the device that kept the temperature at 37 °C and rotated at 50 rpm. The samples (1 mL) were collected at a predetermined time (after 1, 2, 4, 6, 12 and 24 h) (Bayoumi et al. 2022). Vemurafenib absorbance was measured at 307 nm, and the concentration of vemurafenib in each sample was estimated using the calibration curve equation. The cumulative amount released was calculated and divided by surface area to determine the cumulative amount released per area.

After skin permeability as finished, the skin of each ME-based gel (GP1–GP6) was washed with 7.8 phosphate buffer many times to remove any gel on the surface of the skin. The skin was cut into small pieces and immersed in up to 10 mL of methanol and sonicated for a few minutes. The sample was centrifuged at 6000 rpm for 10 min, and the absorbance of vemurafenib was measured spectrophotometrically at 305 nm. Skin deposition was measured using the following equation (Fan et al. 2013):

$Skin deposition (\%)=\frac{Actual drug content}{Theoretical drug content}*100\%$ (3)

Statistical analysis for all experimental data was performed using IBM SPSS Statistic 25 software. Data were expressed as the mean values with their standard deviation (SD). ANOVA with post hoc test was used to approve the significance between results with 𝑃 < 0.05. The DDsolver program was applied to detect the kinetics of drug release.



Pseudoternary phase diagram construction is regarded as a simple and efficient method to predict the effect of different compositions of oil and Smix on the ME system. Fig. 1 represents the pseudoternary phase diagram of different Smix ratios against peppermint oil and water. Fig. 1A–C represent the pseudoternary phase diagram when the Smix ratio used was 1:2, 1:3 and 1:4, respectively. The shaded area in the previously mentioned figure represented the two-phase or macroemulsion area, and the remaining white area represented the one-phase or ME area. The pseudoternary diagrams revealed that the ME area increased when the Smix ratio rose from 1:4 to 1:2. These results indicated that increasing the fraction of SAILs to PEG 400 in the Smix mixture increased the quantity of water held in the ME mixture and increased the ME or one-phase area. These observations agreed with the research, which mentioned that SAILs have superior surface activity to conventional non-ionic surfactants and co-surfactants that result in dramatically decreased surface tension (Ali et al. 2019; Hammodi and Abd Alhammid 2020).

Figure 1. 

Pseudoternary phase diagram. A–C. Represent the pseudoternary phase diagram at the Smix ratios of 1:2, 1:3 and 1:4, respectively.

The average particle size and PDI of all prepared SAIL-based formulas are listed in Table 3. When comparing formulas with the same Smix ratios and Smix % but with differences in the percent of peppermint oil, CP1 had an average particle size of 249.8 ± 3.7 nm, whereas CP 3 had an average particle size of 959.6± 16.9 nm. Similarly, a significant increase (𝑃 < 0.05) was observed in the average particle size between CP6 (that had 15% of peppermint oil and average particle size of 456.6 ± 31.3 nm) and CP4 (that contained 10% of peppermint oil and average particle size of 237.0 ± 3.7 nm). The same trend was found when comparing the particle size of CP7 and CP9. These observations could be explained by the fact that increasing the percentage of oils in the prepared MEs could increase the swelling and size of ME particles (Chrismaurin et al. 2023).

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Increasing the concentration of Smix (while keeping the Smix ratio constant) from 60% in CP1 to 70% in CP2 decreased the particle size from 249.8 ± 3.7 nm in CP1 to 171.3 ± 3.6 nm. The same observation was found with CP4 (with 60% of Smix and average particle size of 237.0 ± 3.7 nm) and CP5 (with 70% of Smix and average particle size of 11.4 ± 1.15 nm). These observations continued when the average particle size of CP7 (with 70% of Smix and average particle size of 58.1 ± 1.3 nm) was compared with that of CP8 (with 70% of Smix and average particle size of 5.2 ± 0.4 nm). Increasing the quantity or percent of Smix in the formulas could decrease the surface tension and dramatically decrease the free energy needed to break down the ME droplets into small particles (Sarheed et al. 2020; Hamed and Abd Alhammid 2021).

Moreover, increasing the quantity of SAILs in the Smix ratio by reserving the Smix percent in the formulas decreased the particle size of the prepared SAIL-based MEs. For example, CP2 (with Smix ratio of 1:4 w/w) had an average particle size of 171.3 ± 3.6 nm, CP5 (with Smix ratio of 1:3 w/w) had an average particle size of 11.4 ± 1.15 nm and CP8 (with Smix ratio of 1:2 w/w) had an average particle size of 5.2 ± 0.4 nm. All these formulas had the same Smix percent of 70%, but they differed in the quantity or ratio of SAILs in the Smix in these formulas. These observations led to the prediction that increasing the ratio of SAILs in Smix could significantly decrease the particle size of the prepared ME droplets and prove the superiority of SAILs in decreasing the interfacial tension and the surface free energy to break the ME droplets to small droplets.

All the prepared SAIL formulas were yellow translucent formulas with no signs of turbidity or separation. In addition, all formulas passed the centrifugation test without any separation, coalescence, or turbidity.

All formulas that had 15% of peppermint oil (CP3, CP6 and CP9) failed to pass the dilution test and showed turbidity when diluted. All formulas with a Smix ratio of 1:4 failed the dilution test (CP1, CP2 and CP3). Only one formula (CP5) with a Smix ratio of 1:3 passed the dilution test. CP7 and CP8 with Smix ratio of 1:2 passed the dilution test. Results from the dilution test are listed in Table 3.

EC is an important test to predict the type of MEs whether they are o/w or w/o type. The o/w MEs, in which water was the continuous phase, had water channels that were capable of carrying electrical current. All the prepared SAIL-based MEs (CP1–CP9) had electrical charge. These observations proved that the prepared MEs were o/w type (Table 3; El Agamy and El Maghraby 2015).

All the prepared SAIL-based MEs (CP1–CP9) had a pH range of 4.51 ± 0.18 to 5.86 ± 0.05. This pH range is acceptable for topical preparations that have a gentle effect on the skin without irritation. The pH of formulas that had 10% of peppermint oil (containing high percentage of Smix), which included formulas CP1, CP2, CP4, CP5, CP7 and CP8, was significantly higher than the pH of formulas with 15% of peppermint oil (containing low percent of Smix), which included formulas CP3, CP6 and CP9 (Table 3).

The drug contents for different SAIL-based MEs (CP1–CP9) are listed in Table 3 and ranged from 99.5 ± 0.3% to 98.9 ± 0.8%. The US Pharmacopeia accepts formulas or dosage forms with drug content percentages ranging from 85% to 115%. Hence, all the prepared MEs had drug contents within the acceptable range (Hammodi and Abd Alhammid 2020).

The release profiles of different prepared SAIL-based MEs (CP1–CP9) are shown in Fig. 2. All formulas (CP1–CP9) showed excellent release profiles with cumulative release percentages within the acceptable range within 24 h.

Figure 2. 

Release profile of vemurafenib from different SAIL-based microemulsion formulas.

The kinetics of the vemurafenib release profile from each SAIL-based ME (CP1–CP9) was instigated using the DDsolver program. All formulas showed a first-order release profile with R² ranging from 0.994 to 0.999. Table 4 shows the fit of the SAIL-based ME (CP1–CP9) to different kinetics of drug release including zero-order, first-order and Higuchi kinetics.

The prepared SAIL-based MEs (CP5, CP7 and CP8) had passed all ME characterization and evaluation tests and had droplet size in the ME range (less than 200 nm), were used to prepare me-based hydrogel using HPMC K15M as a gelling agent using the hot and cold method.

All the prepared hydrogels (GP1–GP6) were yellow, clear and translucent. No signs of creaming or gel separation were detected. The results of the pH and spreadability test of the prepared hydrogels (GP1–GP6) are listed in Table 5. The pH ranged from 5.00 ± 0.11 to 5.43 ± 0.06, which was acceptable for topical preparations with no risk of skin irritation. We observed a significant difference in the spreadability of different prepared gels, which may be attributed to the difference in their viscosity.

Each of the prepared ME-based hydrogels (GP1–GP6) was subjected to different shear rates ranging from 1.32 s^-1 to 1326 s^-1. All the hydrogels (GP1–GP6) showed decreasing viscosity upon increasing shear rate (Fig. 3). These results indicated the pseudoplastic behaviour of the hydrogels (GP1–GP6), which could be explained by a temporary breakdown of the complex structure of the polymer linkage and alliance of the polymer layer in a regular manner (Lim et al. 2015; Ferreira et al. 2016). The gel’s pseudoplastic behaviour facilitated the improved spread on the skin upon application.

Figure 3. 

Rheological behaviour of different microemulsion-based hydrogel formulas.

The cumulative amount permeated per square centimetre versus the time of different ME-based hydrogel formulas is shown in Fig. 4. We compared the vemurafenib flux (Jss) and permeability coefficients (Flaherty et al. 2011; Table 5) of GP1 and GP2 (which were prepared from the same microemulsion CP5). As shown in Table 5, GP1 had significantly higher (𝑃 < 0.05) _Jss and _Kp than GP2. This observation could be attributed to the lower viscosity (2747 ± 106 mP·s at 6 rpm) of the prepared GP1 gel compared with the viscosity of GP2 gel (4613 ± 161 mP·s at 6 rpm). The high viscosity of the gel delayed the release of vemurafenib from the gel base, so the release of vemurafenib from the gel was inversely proportional to the gel viscosity. Similar results were found when comparing the permeability profile of GP3 (24465 ± 105 mP·s at 6 rpm) and GP4 (46098 ± 260 mP·s at 6 rpm) where both of these prepared gels were prepared from the same ME (CP7), as well as when comparing the permeability profile of GP3 (13684 ± 132 mP·s at 6 rpm) and GP4 (34000 ± 117 mP·s at 6 rpm) where these prepared gels were prepared from the same ME (CP8).

Figure 4. 

Cumulative amount permeated per square centimetre versus time of different microemulsion-based hydrogel formulas.

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In addition, J_ss and K_p were affected by the Smix ratio and percent present in the ME used to prepare different hydrogels. Formulas containing a higher SAIL percent had J_ss and K_p when compared with the other prepared gels. Thus, GP5 and GP6 (with SAIL approximately 33% of Smix and % Smix 70% w/w) had significantly higher J_ss and K_p compared with GP1 and GP2 (with SAIL 25% of Smix and % Smix 70% w/w). Similarly, GP6 hydrogel (with SAILs 25% of Smix and % Smix 60 % w/w) had significantly higher J_ss and K_p compared with GP3, even though GP3 had lower viscosity (24465 ± 105 mP·s at 6 rpm) than GP6 (34000 ± 117 mP·s at 6 rpm). This elevation in J_ss and K_p can be explained by the number of SAILs present in GP6 when compared with those in GP3. SAILs acted as permeability enhancers. Hydrophilic SAIL-like C14MIB can break the tight junction present in the skin, thereby increasing the paracellular transport of vemurafenib through skin (Agatemor et al. 2018).

Skin deposition tests were performed as the prepared vemurafenib-based hydrogels was used for topical treatment of skin melanoma. Amongst the tested formulas, GP5 had the highest skin deposition rate (45.4% ± 0.2%; Table 5).