Voriconazole was kindly provided by Global Napi Pharmaceuticals, (Giza, Egypt);Sesame oil and Soyabean oil were obtained from ISO CHEM Co. (Cairo, Egypt); Cremophor RH 40® (Polyoxy 40 hydrogenated castor oil) were obtained from NEROL Co. (Cairo, Egypt); Polyethylene glycol 600 (PEG 600) was obtained from MORGAN chemicals (Cairo, Egypt); Glycerin was obtained from El Gomhouria Co. (Assiut, Egypt); Sodium orthophosphate and di-sodium orthophosphate were obtained from El Nasr pharmaceutical chemicals Co., Egypt ; Methanol (99%) and Isopropyl alcohol (99%) were obtained from El Nasr pharmaceutical chemicals Co., Egypt. All other chemicals used of analytical grade and used without further purification.

The drug calibration curve of voriconazole in each 0f phosphate Buffer (pH 7.4); isopropylalcohol and methanol was found to obey Beer’s Lambert law within the concentration range 5 – 40 µg/mL as shown in Figure 1. The obtained line was analyzed by linear regression. The reciprocal value of the slope represented the procedural constant.

From the pseudo-ternary phase diagrams obtained, it was evident that, the size of the microemulsion region within the pseudo-ternary phase diagrams expanded with the increment of the surfactant concentration within the S/Co blend, from 1:1 to 2:1 and from 2:1 to 3:1. This increment is for the most part due to the interfacial tension’s progressive lowering caused by an increment in surfactant concentration. Comparative to the microemulsion region, the gel region of S/Co blend 3:1 was bigger than that of S/Co blend 2:1, showing the marked impact of the surfactant/co-surfactant (S/Co) weight proportion on the gel phase properties (size and position within the pseudo-ternary phase diagram).

To develop microemulsion formulations for topical delivery of poorly water soluble voricinazole, the optimum oil, surfactant and co-surfactant have to be chosen to confirm the capacity of prepared LLCs gel formulations to solubilize the added amount of voriconazole (0.5 % w/w). The solubility of voriconazole within the different oils, surfactants and co-surfactants are shown in Table 4. The solubility of voriconazole was higher in polyethylene glycol 600 (81.84 mg/ml) and chremophor RH40 (49.68 mg/ml). There were no critical contrasts within the solubility of voriconazole in both sesame oil and soya bean oil. The solubility data demonstrated that the addition of cremophore RH40 and PG to oil led to a different change in drug solubility. The high solubility of voriconazole in surfactant and co-surfactant was advantage to increase its solubility in microemulsions.

The polarized light micrographs (Figure 2) suggested that the formulations F5;F6 and F7 which have streaks texture (Maltes crosses and ribbon like structures) identified as lamellar LLC ¹¹, while the formulations (F1,F2,F3,F4,F8,F9,F10,F11 and F12) appear as black background identified as cubic LLC 22.

FT-IR was carried out to examine the compatibility between voriconazole and other LLCs components. Voriconazole FT-IR spectrum (Figure 3) showed OH stretching at 3250–3050 cm-1, aromatic C-H stretching at 3119 cm-1, aliphatic C-H stretching at 2994-2940 cm-1, and C-N stretching at 1507– 1450 cm-1, and C-F stretching at 1586–1473 cm-1, respectively. The given results are in accord with those recorded by khare et al 23. The spectrum of pure cremophor RH 40 showed the absorption band of the OH group, which was expressed as a broad band at 3436 cm-1. The aliphatic C-H bond was seen as a biforked band with two apices at 2925 and 2858 cm-1. The peak carbonyl group of the ester was found at 1734 cm-1. The ether C–O–C stretching was manifested as a strong broad band at 1111 cm-1 24.

The spectrum of soyabean oil and sesame oil showed the absorption band of the OH group, which was expressed as a broad band at 3436 cm-1. The aliphatic C-H bond was seen as a biforked band with two apices at 2925 and 2858 cm-1. The peak carbonyl group of the ester was found at 1746 cm-1 25, 26.

The main characteristic peaks of glycerin were observed at 3385 cm-1 (OH stretching) and 2886 cm-1 (C-H stretching) 27, while main characteristic peaks of PEG 600 observed at 3416 cm-1(OH stretching) and 2876 cm-1(C-H stretching) 28. It was observed that there was no remarkable change in the peaks of pure drug and LLCs components. Hence, no specific interaction was observed between the drug and the components used in the formulations.

Lyotropic liquid crystals droplet size significantly affects permeation of drug into deeper layers of skin, where small droplet size makes it an excellent carrier for promoting drug percutaneous uptake as the number of droplets that can interact on a fixed area of SC will increase when the particle size decreases.

Table 5 shows the mean droplet size, PDI, zeta potential of the selected LLCs gel formulations. All formulations possess small droplet size in the nano range (below 50 nm), except F2 and F7 (63.27 and 66.9 nm, respectively). The small size was expected due to the presence of co-surfactant molecules and the surfactant, which reduces interfacial tension and thus decreases the radius of droplets 29. Formulations F2 and F7 showed a significantly larger mean droplet size compared to other formulations (P < 0.05). This could be attributed to their higher oil concentration compared to other formulations. Marco et al., reported that, there are linear relationship between oil content and droplet size of the prepared microemulsions 30.

The influence of the different ratios of surfactant (Cremophor RH40) to co-surfactant (glycerin or PEG 600) on the droplet the ratio of surfactant to co-surfactant increased from 1:1 to 2:1 (when use glycerin as co-surfactant) and from 2:1 to 3:1 (when use PEG 600) as co-surfactant. This may be attributed to the increased proportion of surfactant which further produce more size reduction 30, 31.

PDI results showed that all of the formulations had droplets in the nano range. The homogeneity of droplet size within the formulation is shown by the PDI, which is the ratio of standard deviation to mean droplet size. The uniformity of the droplet size in the formulation decreases with increasing PDI ³². PDIs value ranged from 0.098 to 0.240 which shows the narrow distribution of droplet size.

Also, Zeta potential of the formulations was measured for determination of droplet charge and/or electrostatic repulsion. The physical stability of any dispersed systems said to increase with the increase in the electrostatic repulsion energy, which is directly proportional to the particle surface charge and the thickness of the diffusion layer ³³. Due to the presence of a non-ionic surfactant, whose diffusive border has an almost neutral charge and supports the stability of the microemulsion, zeta potential values ranged from -5.5 ± 0.40 to -16.2 ± 0.72. The stability of the LLCs gel system is indicated by a negative zeta potential. According to this finding, all formulations contain sufficient charge and mobility to reduce droplet aggregation ³⁴. Uniformity of the droplet size in the formulation decreases with increasing PDI ³², PDIs value ranged from 0.098 to 0.240 which shows the narrow distribution of droplet size.

Table 6 shows pH values of all selected LLCs gel formulations. Since the pH of therapeutic substances applied topically from 5.5 to 6.5, the pH values of the prepared LLC gel could be counted in the tolerable pH range 35.

Good uniformity of drug content among the prepared gel formulations were observed within range from 96.80 ± 1.55 to 102.3 ± 1.26 % w/w (Table 7). These results indicated that the process employed to prepare gels in this study was capable of producing gels with uniform drug content and minimal content variability. Also, drug contents of all formulations were found within limit (90-110 % w/w).

The rheological status of a semisolid drug carrier system is a very important physical parameter. All the LLC gel formulations (Figure 4) exhibited a shear thinning flow (pseudo plastic flow) as the viscosity decreased with increasing the shear rate. This result was in agreement with the findings of Savic et al. ³⁶, and Qian et al. ³⁷, who found that the rheological behavior of liquid crystalline gel phases was plastic or pseudoplastic flow behavior, a property desirable for viscous liquid dispersions or semisolids. All the selected LLCs gel formulations have suitable viscosity for topical application. Formation of LLC ordered structures is responsible for the increased viscosity of the systems ¹¹. Rheological profile of the prepared gel formulations revealed that the formulations with higher surfactant: co-surfactant ratio were more viscous than formulations with lower surfactant: cosurfactant ratio as the following order (3:1 ˃2:1˃1:1), this may be attributed to increased concentration of surfactant (Cremophor RH40). It is also observed that gel formulations with glycerin cosurfactant have higher viscosity than formulations with PEG 600 as cosurfactant.

Spreadability is the term expressed to denote the extent of area to which the gel readily spreads on application to the skin. The spreadability of topical preparations is an important criterion for standardized and simple application on the skin. The efficacy of the gel or the bio-availability efficiency of the formulation depends on the spreadability value. The higher the value of the gel spreadability, the higher is the absorption area and the higher is the bio-available efficiency of the formulation.

Spreadability test was carried out for all LLCs gel formulations. The results (Table 8) showed that for all formulations, the increase in gel viscosity resulted in to lower spreadability values. These findings were in agreement with Kishor et al. ¹² who reported that, the increase in viscosity of gel formulations resulted in poor spreadability values. From the Table 8, it was observed that, formulations F1, F2, F9, F10, F11 and F12 gives higher spreadability values than other formulations, these results may be due to lower viscosity values of these formulations.

The release patterns (Figure 5) revealed that, drug release rate from various LLC gel formulations was significantly slower than that from carbopol gel containing 0.5 % w/w voriconazole (P< 0.001), this ensure that LLCs gel formulations provide sustained release effect. Carbopol gel showed more than 50 % release of voriconazole within first 4 hr whereas the LLCs gel formulations released less than 20 % of voriconazole within first 4 hr. Voriconazole release was successfully prolonged due to the previously discussed high viscosities of LLC gel formulations. Results demonstrated that all LLCs gel formulations caused a sustained drug release up to 24 hours. Makai et al. ³⁸ reported the ability of LLCs systems to prolong the release of water soluble and water insoluble drugs. Also, sustained release profile of gel formulations may be attributed to organized internal structure) and high viscosity of lyotropic phase. LLCs gel formulations can be a promise candidate for topical delivery of voriconazole, since the high viscosity of LLC phases and its more prolonged release profile may provide sustained skin concentrations of voriconazole for a prolonged time. These characteristics can improve patient compliance apart from reducing the frequency and severity of voriconazole side effects. LLC gel formulations containing PEG 600 as co-surfactant (F9, F11 and F12) have higher drug release % than LLC gel formulations containing glycerin as co-surfactant, this may be attributed to lower viscosity values of gel formulations containing PEG 600 as cosurfactant. This result was in agreement with the findings of Singh et al. ⁷, who found that release rate of metronidazole increased with lower viscosity of liquid crystalline systems. Another explanation is higher solubilization capacity of PEG 600 than glycerin, the equilibrium solubility of voriconazole in PEG 600 is (81.84 mg/mL) while in glycerin is (4.52 mg/mL), Prasanna et al. ⁶, reported that microemulsions with PEG 600 as cosurfactant had shown slightly enhanced voriconazole release compared to microemulsions which contain propylene glycol as cosurfactant at same S/Co ratios. El-Hadidy et al. ³⁹ reported that prepared microemulsions using glycerol as the co-surfactant, showed significantly smaller voriconazole release values than MEs using sorbitol as co-surfactant, this retardant effect could be related to the higher viscosity of MEs contains glycerol. When comparing LLCs gel formulations containing the same components except for type of oil (F9 vs F10) and (F11 vs F12), it is clear that oil type has no significant effect on release rate of voriconazole. It may be attributed to that voriconazole has approximately same solubility in both two oils (equilibrium solubility of drug in sesame oil and soyabean oil is 7.37 mg/mL and 8.24 mg/mL respectively). As presented in Figure 6, The release of voriconazole from different LLC gel formulations after 24 hr can be ranked in the following descending order: (F12 ˃ F4 ˃ F9 ˃ F11˃ F3, F5, F8 ˃ F10 ˃ F1 ˃ F6 ˃ F2 ˃ F7).

Table 9 showed the results of fitted voriconazole release data from different LLC gel formulations to different kinetic models. The drug release from the gel formulations followed first order model except F9. The diffusion exponent (n) of Korsmeyer-peppas equation was ˃ 0.89 which indicate super case II mechanism (swelling controlled).

Ex-vivo permeation of selected formulations F3, F4, F5 (Figure 6 A) F11 and F12 (Figure 6 B) were studied and carbopol gel containing 0.5 % w/w voriconazole was used as control. Figure 6 shows the cumulative amount permeated of voriconazole through skin from various formulations as function of time. It was found that significant (p < 0.05) increase in amount of voriconazole permeated from all LLC gel formulations compared to that permeated from carbopol gel (0.5 % w/w voriconazole). Several mechanisms have been proposed to explain the penetration-enhancing effect of LLC gel. Most likely, it is the overall combination of various mechanisms that result in the penetration-enhancing effect. The first possible mechanism was related to the similarity of LLC structures (lamellar and cubic phases) to the structure of skin. Liquid crystal-based formulation exhibits high hydration and easy distribution due to the similarity in the structures of LC and stratum corneum.

The cubic phase system exhibits strong bio-adhesive property and forms a biological membrane like structure over the skin on topical application 40, 41. Also, mesophases with a lamellar structure demonstrate great similarity to the intercellular lipid membrane of the skin ³⁸. The second possible mechanism is the penetration enhancing effect of LLC gel components. Through the use of a nonionic surfactant (Cremophor RH 40), a reversible increase in the permeability of the stratum corneum can be achieved without irreversible skin irritation. The LLC systems exhibit good penetration, due to the very low interfacial surface tension arising at the oil/water interface, and they may facilitate the progressive diffusion of biologically active substances into the skin or systemic circulation ³⁸. The third possible mechanism is the small nano oil droplets which provide a larger surface area for permeation of drug in to the skin ⁴².

When comparing LLCs gel formulations containing the same components except for type of oil (F11 vs F12), it’s clear that oil type has no significant effect on skin permeation of voriconazole. It may be attributed to that drug has approximately same solubility in both two oils. Also, the results showed that formulation F3 containing S/Co ratio of 2:1 achieved a significant (P< 0.05) increase in skin permeation effect compared to formulation F5 containing S/Co ratio of 3:1. This may be attributed to higher viscosity of F5. This finding was not in accordance with results obtained by F4 which contain S/Co ratio of 2:1. The amount of voriconazole that permeate through skin (Q) was calculated and plotted against time. The slope of linear section of the figure was used to compute the transdermal drug flux (Jss) and the apparent permeability coefficient (Papp) was determined through Jss / Co equation where Co is the initial drug concentration ³⁴ as shown in Table 10.

Table 10 showed that from all LLC gel formulations studied, formulations F3 and F12 provided fluxes higher than the rest of the formulations (F4, F5, F11). Also, F3 and F12exhibited higher permeability values for voriconazole. Both formulations (F3 and F12) were selected for further evaluation.

Skin deposition of the drug from different formulations was also investigated (Figure 7). The amount of drug deposited in skin from carbopol gel (0.5 % w/w voriconazole) was significantly lower than that from any of the LLC gel formulations (p<0.05). This enhanced skin deposition for the drug loaded into the LLC gel might be attributed to the enhanced penetration of the drug into the skin layers and structural similarity of the LLC structures with skin ⁴³.

The amount of the drug deposited in skin after 24 hr was significantly higher (P< 0.05) for LLC gel formulations F3 and F12 when compared with F4, F5 and F11. So based on skin deposition and skin permeation results, F3 and F12 were selected for further evaluation.

Rhodamine B (log P: 1.95) was chosen as model compound which have similar lipophilic features with voriconazole (log P: 1.8) and loaded into optimized LLC gel formulations (F3 and F12) to visualize their penetration ability into skin layers ¹⁶. The skin penetration enhancement effect of LLC and the localization of model fluorescent marker following the topical application of LLC gel formulation was visualized using CLSM.

The confocal images in Figure 8 showed that the LLC gel were able to deliver fluorescent dye to all skin layers at the end of 24 hr. These results were consistent with those of in-vitro skin permeation and deposition study. Taken together, CLSM images further confirm that LLC gel may significantly improve drug permeability and retention.

Rhodamine B dermal deposition concentrations are estimated based on fluorescent intensity in skin layers which were quantitatively analyzed by Image-J software ⁴⁴. As shown in Figure 8, the results obtained revealed that LLC formulations F3 and F12 Provide approximately the same Rhodamine B concentrations (fluorescence intensity) in all skin layers with no significant difference between the two formulations (F3 and F12).

The results suggest that the proposed formulations (F3 and F12) were able to deliver voriconazole up to and beyond the epidermis which can be suitable for topical fungal infection control. LLC gel formulation cause adherence to the stratum corneum followed by increased voriconazole penetration into viable layer of the skin (The extent of penetration into deeper skin layers at the depth up to 766 µm for F2 and up to 1079 µm for F12).

Although all the materials used for preparation of LLC gel fell under the Generally Regarded as Safe (GRAS) category, concentrations of all materials are a very critical issue for these formulations. For example, a large amount of surfactants is usually an irritant to the skin. Therefore, a skin irritation test was performed to confirm that the concentration of materials used for LLC gel preparation was safe. The results are shown in Table 11 and Figure 9. Draize et al. ⁴⁵ mentioned that a value of the primary irritancy index (PII) < 2 indicates that the applied formulation is a non-irritant to human skin. Therefore, F12 and F3 were considered to be a non-irritant as PII was < 2. However, animals treated with 0.8% w/v aqueous formalin as standard irritant demonstrated PII = 6.43 ± 1.6 indicating high irritancy. Negative control group indicated no irritation as no medication was applied on to the skin. Therefore, on the basis of outcomes of this study, formulation can be declared as non-irritant and safe for skin application.

The agar diffusion method was performed to determine the effectiveness of the prepared LLC formulations with respect to carbopol gel (0.5 % w/w voriconazole) against different species of fungi including Candida albicans, Aspergillus flavus and Aspergillus niger. The antifungal activities were measured as diameter (mm) of zone of inhibition.

The results showed that the diameter of zone of inhibition obtained from LLC gel formulations (F3 and F12) was significantly higher than that of carbopol gel containing 0.5 %w/w voriconazole (P<0.05) while plain LLC gel formulations (F3 and F12) did not show any activity against all the examined strains of fungi as observed in Figure 10. These results indicating the superior antifungal activity efficacy of LLC gel formulations over carbopol gel containing 0.5 % w/w voriconazole. The enhanced in-vitro antifungal activity of LLC gel formulations may be attributed to enhanced penetration of oil globules containing voriconazole through fungal cell walls to inhibit ergosterol synthesis ⁴⁶. In addition, sustained release of voriconazole from LLC can control the growth of fungi for longer duration than carbopol gel (0.5 % w/w voriconazole) ². From Figure 10, it was clear that, the inhibition zone of voriconazole against A. flavus and A. niger was significantly higher than that of C. albicans. This was in agreement with the results obtained by Neslihan et al. who illustrated the significant activity of voriconazole against Aspergillus species ⁴⁷.

Evaluation of antifungal activity using Candida albicans is widely used. Before induction of superficial fungal infection, all animals showed normal skin structure without any clinical features of fungal infection, for example inflammation, edema, cracking, or color changes ⁴⁸. After fungal infection induction, the animals were observed on daily basis for signs of infection. The topical treatment was started after 7 days, after the appearance of fungal infection signs (more intense redness, inflammation, scales, ulcers and edema) and confirmation of fungal infection on the skin of animals using light microscope (Figure 11). After continuous application of different treatments once daily for seven days: Negative control group showed normal skin without any sign of infection and no histological changes. On the other hand, positive control group presented slight decrease in skin elasticity with inflammation, ulcers and scares which confirmed by using histological image with necrosis and inflammatory cells infiltration in the dermis.

In groups treated with LLC gel formulations (F3 and F12), fungal infection signs were significantly disappeared at day 5 and treatment was continued for another two days to ensure eradication of fungal infection as shown in Figure 12, while fungal infection signs (redness and inflammation and scales) were still present in groups treated with plain LLC formulations and carbopol gel (0.5 % w/w voriconazole).

The skin that had been treated with LLC gel (F3 and F12) seemed healthy and smooth, with no signs of infection like redness, swelling or scarring. These dramatic shifts suggest that LLC gel showed therapeutic effects on Candida skin infections, and these results were supported by histopathological examination.

Rats in negative control group had intact epidermis covered with normal keratin layer and dermis containing normally distributed skin adnexa (Figure 13 A). Histopathological examination of skin from Candida ablicans-infected rats showed coagulation necrosis of sub-epidermal with formation of micro-abscesses at the dermis-epidermal junction (Figure 13 B), hyperkeratosis and acanthosis (Figure 13 C). The dermis and subcutis revealed mononuclear cellular infiltration and loss of skin adnexa (Figure 13 D).

On the other hand, treatment of Candida ablicans-infected skin with F12 formulation after 7 days of infection for 7 days resulted in formation of a thin epidermal layer covering the skin and absence of the mononuclear cellular infiltration in the dermis and subcutis (Figure 13 C). As well, treatment of Candida ablicans-infected rats with F3 formulation leads to formation of a well-developed epidermal layer on the skin and absence of the mononuclear cellular infiltration in the dermis and subcutis (Figure 13 D).

Similar treatment of infected rats with carbopol gel showed an improvement of skin histology except for denuded areas of epidermis alternated with areas of a canthosis and hyperkeratosis (Figure 13 G).

Treatment of infected rats with either F12 plain or F3 plain caused no histological improvement in both epidermis and dermis. Animals showed loss of epidermal layer and degenerative changes of skin adnexa, and coagulation necrosis of sub-epidermal tissues (Figure 13 E and F), respectively.

The formulations were found to be stable for six months at room temperature and at 4-8 ºC. Table 12, Table 13 show that, there was no change in appearance (color and turbidity), no phase separation, no drug precipitation. Also, there was no significant change in pH, drug content, and viscosity. This was expected due to the thermodynamic stability of LLC gel system. However, other properties were slightly altered such as pH values of formulations (F3 and F12) as well as droplet size. The slight pH decrease of the two formulations may be attributed to greater number of hydrogen ion liberated from hydrolytic degradation of both oils (sesame and soyabean oils)49. On the other hand, the increase droplet size contributed to reduction in the zeta potential from -7.53 to -3.4 for F3 and from -7.46 to -4.47 for F12. Also, the reduction in zeta confirms liberation of hydrogen ion, which carry positive charge led to decrease negative charge (zeta potential) in the system.

The authors would like to express their gratitude and appreciation to Prof. Dr. Sary Khaleel Abd Elghaffar Nasr faculty of Vetrinary Medicine, Pathology Department, Assiut University, Assiut, Egypt. For the interpretation of the Histopathological data.

This research did not receive any specific grant from funding agencies in the public, commercial and not for profit sectors.

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

The authors declare that they have no known competing financial interests or personal relationships that have appeared to influence the work reported in this article.

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