3.1. Microemulsion Systems
Development of a microemulsion containing the poorly water-soluble drug, sildenafil, requires ingredients that exhibit excellent drug solubilization. A previous study prepared sildenafil (base) microemulsions, and the results revealed that the solubility of sildenafil was considerably higher (by approximately 10 times) in oleic acid (75.5 ± 11.3 mg/mL) than that of sildenafil citrate in the present study. However, the solubility of sildenafil (base) in surfactants showed the opposite trend [
21]. The water solubility of sildenafil and sildenafil citrate was reported to be 0.0 ± 0.0 mg/mL and 3.20 ± 0.11 to 4.1 ± 1.3 mg/mL, respectively [
5,
22]. Sildenafil citrate exhibits a higher water solubility compared to the sildenafil base; however, sildenafil citrate requires oil to improve its permeability through the skin. The higher solubility of sildenafil (base) in oil than sildenafil citrate in oil can be attributed to the polarity of the citrate salt and the low oil partition coefficient of the salt form. However, because the objective of this study was to load sildenafil citrate in microemulsion systems together with a hydrogel to develop a new formulation, sildenafil citrate was used.
The saturated solubility study of sildenafil citrate in various oils, surfactants, and co-surfactant is shown in
Table 2. The highest solubility of sildenafil citrate was obtained with oleic acid (7.63 ± 0.48 mg/mL), followed by isopropyl myristate, while the lowest solubility was obtained with castor oil. The association between the weak basic drug sildenafil (pKa 8.7) and the acidic oleic acid could be responsible for the highest solubility in this oil. These results are similar to those of a previous report by Elshafeey et al. [
5] and Jung et al. [
22], although the solubility values are different. The experiments on the solubility of sildenafil citrate in different surfactants demonstrated the highest solubility in Tween 80 among all the surfactants tested, followed by Cremophore RH40. These results contrast with the previous report by Elnaggar et al. [
23], which placed Cremophore RH40 higher than Tween 40 in terms of sildenafil citrate solubility. In the case of co-surfactants, PEG400 and propylene glycol exhibited the maximum solubility of sildenafil citrate (5.16 ± 0.22 and 4.10 ± 0.58 mg/mL, respectively). Considering the solubility of sildenafil citrate, isopropyl myristate and oleic acid should be the most appropriate oils for developing microemulsions as the solubility of sildenafil citrate in isopropyl myristate and oleic acid is 7.63 ± 0.48 and 4.21 ± 0.65 mg/mL, respectively. Thus, both oleic acid and isopropyl myristate are considered as suitable oil phases due to the high drug solubility and low risk of skin toxicity. Therefore, the surfactant and co-surfactant systems selected were Tween 80, PEG400, and propylene glycol. Oleic acid was used as the oil phase to construct the phase diagram for the microemulsion systems because it was a common absorption enhancer in transdermal excipients and possesses the suitable properties of surfactants and co-surfactants.
The pseudo-ternary phase diagrams of the microemulsions were constructed using isopropyl myristate or oleic acid as the oil phase. Tween 80 was used as a surfactant and PEG400 or propylene glycol was used as a co-surfactant at a surfactant-to-co-surfactant ratio of 1:1, 1:2, 1:3, 2:1, or 3:1. The pseudo-ternary phase diagrams obtained from these systems are displayed in
Figure 2.
The construction of a pseudo-ternary phase diagrams helps in determining the concentration range of the components to check for the existence of microemulsions. Microemulsion systems are formed at room temperature. When water was added to the selected oil mixtures (a mixture of isopropyl myristate with Tween80/PEG400 or a mixture of oleic acid with Tween 80/propylene glycol) in all formulations, a continuous transition from water-in-oil systems (W/O) to oil-in-water (O/W) systems was observed, and a transparent, one-phase, and low-viscosity system was obtained. The O/W microemulsions formed are shown in a three-component triangular diagram. The transparent to translucent microemulsion region is shown in phase diagrams (
Figure 2). No distinct phase inversion of the microemulsions was noted. The remaining region on the phase diagram represents the turbid and conventional emulsions based on visual observation. The incorporation of isopropyl myristate and Tween 80/PEG400 as the surfactant and co-surfactant in different ratios is shown in
Figure 2a. When the maximum amount of Tween 80/PEG400-incorporated water was used in the oil-surfactant system, a microemulsion zone was formed. As the ratio of the surfactant-to-co-surfactant increased, the existence area of the microemulsion increased, reaching a maximum at 2:1. The addition of a surfactant and co-surfactant mixture in a 2:1 ratio increased the water incorporation to a maximum of 40% compared to 2% in the co-surfactant-free system.
The pseudo-ternary phase diagram of the oleic acid system obtained using Tween 80/propylene glycol as the surfactant and co-surfactant is shown in
Figure 2b. The results were similar to those of the isopropyl myristate system. In addition, when the ratio of Tween 80 was low, the microemulsion area was small. This result indicates that the surfactant plays an important role in the microemulsion formation in this system.
3.2. Sildenafil Citrate Microemulsion-Loaded Hydrogel
Various microemulsion systems were selected from the phase diagram of
Figure 2 for all ratios of surfactants and co-surfactants. The parameters of the microemulsions are shown in
Table 3. All the microemulsions had small average droplet diameters ranging from 30 to 600 nm. The polydispersity index showed that all the microemulsions had a narrow size distribution. However, when sildenafil citrate was added to the microemulsion in large amounts (more than about 10%), a milky white liquid was obtained.
The composition of the microemulsion system used to prepare the microemulsion is shown in
Table 3, and the solubility of sildenafil citrate in the microemulsion system was measured. In the isopropyl myristate system, the microemulsion contained 30% isopropyl myristate, 40% Tween 80, and 20% PEG400, and showed the lowest droplet size at 30 ± 6 nm and the highest sildenafil citrate solubility at 122 ± 28 mg/mL. Furthermore, this ratio contained a high content of Tween 80 (40–43%), which is used as an enhancer for transdermal delivery [
24]. In the oleic acid system, the microemulsion contained 22% oleic acid, 43% Tween 80, and 22% propylene glycol gave the lowest droplet size of 320 ± 45 nm with the highest sildenafil citrate solubility of 128 ± 8 mg/mL. Notably, the solubility of sildenafil citrate in the microemulsion system was improved. Sildenafil citrate solubility reached approximately 4.21–7.63 mg/mL in isopropyl myristate, a 1.3–2.4-fold increase compared with its intrinsic solubility in water (3.20 ± 0.11 mg/mL) [
5] and 38–40-fold increase compared to its solubility in the microemulsion of the isopropyl myristate or oleic acid systems (122 ± 28 and 128 ± 8 mg/mL), respectively. Thus, the composition ratios of 30:40:20:10 for isopropyl myristate:Tween 80:PEG400:water and 22:43:22:13 for oleic acid:Tween 80:propylene glycol:water were chosen for the isopropyl myristate-based microemulsion system and oleic acid-based microemulsion system, respectively. Notably, the solubility of sildenafil citrate in the oleic acid-based microemulsion was higher than that in the isopropyl myristate-based microemulsion system.
Based on these results, microemulsions containing 12% sildenafil citrate were prepared at a surfactant-to-co-surfactant ratio of 2:1. The microemulsions containing sildenafil citrate resulted in phase diagrams similar to those of the microemulsions without the drug. The detailed compositions of the three microemulsions are shown in
Table 1, with the same composition as that shown in
Table 3. All these formulations existed inside the area of microemulsion formation, thereby forming a clear microemulsion at the additive concentrations examined. Hydrogel-thickened microemulsions were formulated by mixing poloxamer 188 solution with the microemulsion.
A photograph of the microemulsion of both the isopropyl myristate-based and oleic acid-based systems are shown in
Figure 3. Microemulsions were clear, transparent, and had a low viscosity (
Figure 3A,E). When 12% sildenafil citrate was added to the microemulsion, a milky white opaque microemulsion was obtained (
Figure 3B,F). When poloxamer 188 was added to microemulsion in order to thicken the system without sildenafil citrate, the microemulsion-loaded hydrogel remained clear and transparent with the increasing formulation viscosity (
Figure 3C,G). Finally, sildenafil citrate (12%) and poloxamer 188 were added to the microemulsion; consequently, highly viscous and milky white hydrogels were obtained (
Figure 3D,H). The opacity of the sildenafil citrate microemulsion-loaded hydrogel occurred even when they were dissolved, but the presence of an off-white powder of sildenafil citrate caused a milky white emulsion and increased the particle size of the systems.
The physical properties of the sildenafil citrate microemulsion systems with and without poloxamer 188 are summarized in
Table 4. The droplet size of the sildenafil citrate microemulsion-loaded hydrogel ranged from 461 ± 108 nm to 619 ± 68 nm, which was slightly higher than that of the microemulsion without poloxamer 188 (450 ± 120 nm to 525 ± 45 nm). This is caused by the swelling of the microemulsion in the hydrogel system. The pH of the sildenafil citrate microemulsion-loaded hydrogel of both formulations was 5.27 ± 0.04 and 4.67 ± 0.07 for the isopropyl myristate and oleic acid systems, respectively, compared to 5.14 ± 0.02 and 4.54 ± 0.03 for the formulation free of a gelling agent. These results indicate that poloxamer does not affect the pH of the formulation. At this pH range, the formulation the microemulsion system leads to less irritation when applied to the skin of the penis, especially the glans penis, which is sensitive to response.
The viscosity of the sildenafil citrate isopropyl myristate-based microemulsion was 4569 ± 79 mPa s, and that of the sildenafil citrate oleic acid-based microemulsion was 1256 ± 55 mPa s. Increasing viscosity was obtained with both the sildenafil citrate microemulsion systems when poloxamer 188 was added to the formulations. The viscosity of the isopropyl myristate-based and oleic acid-based microemulsion was 11,997 ± 1465 mPa s and 3231 ± 69 mPa s, respectively. The viscosity of the microemulsion was significantly increased by adding poloxamer 188 to the hydrogel.
Spreadability of the isopropyl myristate-based microemulsion-loaded hydrogel was lower than that of the oleic acid-based microemulsion-loaded hydrogel system because of the higher viscosity of the isopropyl myristate-based formulation. Spreadability is a particularly important parameter, as it shows the behaviour of the microemulsion-loaded hydrogels and the ease with which the formulation is applied to the glans penis.
3.3. FTIR Analysis
The IR spectra in the frequency region from 400 to 4000 cm
−1 for sildenafil citrate and for the microemulsion-loaded hydrogels with and without sildenafil citrate (with both the isopropyl myristate and oleic acid-based systems) are shown in
Figure 4, and the interpretation is shown in
Table 5. The asymmetric (~1255 cm
−1) and symmetric (~1323 cm
−1) SO
2 stretching bands associated with the molecular vibration of the sulphone group are shown in
Table 5, and are similar to that reported in previous studies [
25,
26]. After the sildenafil citrate dissolved in the microemulsion, both the asymmetric and symmetric SO
2 stretching bands were absent. The IR spectra of the microemulsions of both the isopropyl myristate- and the oleic acid-based systems showed a broad and strong band in the range 3300–3500 cm
−1 (owing to O–H stretching of the gel). The peaks centred around 2850–2950 cm
−1 arise from the C–H stretching peaks of all samples. The bands at 1632 and 1640 cm
−1 are the so-called N–H bend bands of amide in the sildenafil microemulsion of the isopropyl myristate and oleic acid-based systems, respectively. The band shift to higher wavenumbers of sildenafil citrate from 1618 cm
−1 to 1632–1640 cm
−1 led to the bending of the secondary amide in the cyclic amide, which nearly overlapped with the rich C=O stretching band of the microemulsion systems [
27]. The band at 1589 cm
−1 in the spectrum can be attributed to the symmetric stretching frequency of the COOH groups belonging to the citrate ion [
25] appearing in the isopropyl myristate-based system, but not in the oleic acid-based system.
3.4. Rheological Analysis
In this study, poloxamer 188 was selected because it is commonly used in pharmaceutical preparations and is readily soluble in water [
28]. Poloxamer 188 solution (10%) was added to the microemulsion system to prepare the microemulsion-based hydrogel after dissolving sildenafil citrate into the microemulsion system. However, after poloxamer 188 completely swelled in the microemulsion, it still exhibited a transparent and clear characteristic. We found that poloxamer 188 increased the viscosity of the microemulsion, maintained the microemulsion structure, and was a good matrix that swelled in the microemulsion system.
The viscosity and elasticity of the hydrogel samples can be monitored using the parameters of elastic modulus (G′) and loss modulus (G″) [
29]. The viscous liquid or elastic hydrogel shows that G′ is smaller than G″. Furthermore, a larger G′ reflects the inherent characteristics of the solid samples, and the phase transition from liquid to semisolid. The rheological properties of the microemulsion-loaded hydrogel with and without sildenafil citrate are shown in
Figure 5. The sildenafil citrate microemulsion-loaded hydrogel showed a shear thinning system.
The temperature sweep plots of the hydrogels are shown in
Figure 5. The behaviour of the two hydrogels was significantly different, indicating a difference in their structure. For the hydrogel prepared from the isopropyl myristate-based microemulsion system, throughout the temperature sweep, both during heating and cooling, G′ was higher than G″ at temperatures below 18 °C, and the values of both moduli were larger than those of the hydrogels prepared from the oleic acid-based microemulsion system.
The isopropyl myristate microemulsion hydrogel exhibited a thermo-reversible nature; the gel showed breaks near 18 °C and remained in the sol phase at higher temperatures. The hydrogel retained its sol form after cooling to a temperature of 18 °C. These results suggest that crosslinks are responsible for the gel breaks at higher temperatures for the hydrogel prepared at a neutral pH, and the hydrogel remains stable only at low to moderate temperatures.
3.7. In Vitro Skin Permeation Studies
The permeation rates of the sildenafil citrate-loaded hydrogel from the microemulsion across the membrane from the microemulsion-loaded hydrogel are shown in
Figure 6, and the skin permeation parameters are shown in
Table 7. The isopropyl myristate-based microemulsion system showed the highest permeation rate (2.105 ± 0.003 μg/cm
2/h), followed by sildenafil citrate suspension (1.070 ± 0.177 μg cm
−2 h
−1) and oleic acid-based microemulsion system (0.984 ± 0.244 μg/cm
2/h). The isopropyl myristate-based microemulsion system showed the highest permeation rate of this drug. This may be attributed to the permeation-enhancing effect of isopropyl myristate, Tween 80, and PEG400, with a lower droplet size in the microemulsion. Although isopropyl myristate, oleic acid, and Tween 80 have also been frequently used as powerful permeation enhancers, isopropyl myristate acts as a more effective permeation enhancer for topical delivery in microemulsion systems [
30].
The skin fluxes of sildenafil citrate from the isopropyl myristate-based microemulsion system were two times higher than those of the oleic acid-based microemulsion system and sildenafil citrate suspension. This result might be attributed to the considerably higher solubility and diffusion rates of sildenafil citrate from the microemulsions as a liquid medium than those from the sildenafil citrate solution without any enhancer. Statistical comparison of the flux throughout 24 h showed that the isopropyl myristate-based microemulsion system provided fluxes (
p < 0.05) higher than those of the saturated solution of sildenafil citrate and oleic acid-based microemulsion system. The permeation profiles of the sildenafil citrate microemulsions followed zero-order release kinetics. A possible explanation is that the surfactant and co-surfactant may exist in each phase of the microemulsion; hence, the active ingredient can be partly solubilised in the external phase, and the depletion of the active ingredient in the external phase because of the permeation into the skin can be supplemented by releasing the active ingredient from the internal phase. Further, the zero-order release kinetics and sustained, controlled, and prolonged delivery of active ingredient were obtained. Oleic acid and propylene glycol as permeation enhancers had a strong permeation-enhancing effect and could enhance the solubility of sildenafil citrate in the skin. The partition coefficient could be increased owing to permeation enhancers. In addition, because of the small droplet diameters of the microemulsions, the likely mechanism may also be the permeation of sildenafil citrate directly from the droplets into the stratum corneum without microemulsion fusion to the stratum corneum and the subsequent permeation enhancement. The decrease in oleic acid release rate compared to that of the control can be attributed to the mechanism of fatty acids, such as partitioning into lipid bilayers, i.e., the stratum corneum, and the formation of lipophilic complexes with the drugs [
31].
3.9. Drug Metabolism
The accumulation of drugs after the pure sildenafil citrate suspension and sildenafil citrate microemulsion-loaded hydrogel (isopropyl myristate system) were incubated with HepG2 cells is shown in
Figure S3 (see
supplementary files). The sildenafil citrate microemulsion-loaded hydrogel could penetrate cells at a 6 times higher rate than that of the sildenafil suspension.
The LC-MS/MS chromatogram of sildenafil citrate (pure drug) and the drug extracted from the supernatant of the HepG2 cells incubated with the sildenafil citrate microemulsion-loaded hydrogel or sildenafil aqueous suspension for 6 h is shown in
Figure S4 (see
supplementary files). Pure sildenafil citrate and sildenafil citrate extracted from the HepG2 cell supernatant incubated in suspension showed similar results. However, different mass results were obtained from the sildenafil citrate extracted from the HepG2 cell supernatant incubated with the sildenafil citrate microemulsion-loaded hydrogel. The proposed drug metabolism pathways of the sildenafil citrate aqueous suspension and sildenafil citrate microemulsion-loaded hydrogel are shown in
Figure 7. The sildenafil citrate pure drug suspension induces the following metabolic pathways: piperazine
N-demethylation; pyrazole
N-demethylation
N,
N-deethylation (
m/z 299, 283, 377, 311); and aliphatic hydroxylation [
32]. This result contrasts with the sildenafil citrate microemulsion-loaded hydrogel showing mono-hydroxylation (
m/z 491) of sildenafil citrate, which is a major metabolite of over 90% of the parent compound. The metabolism of the sildenafil citrate microemulsion-loaded hydrogel was concentration-dependent; consequently, when a high drug concentration was applied, the metabolism was saturated. In addition, the metabolite of the sildenafil formulation was decreased by approximately 50% compared to that of the sildenafil suspension.