Development and Optimization of Luliconazole Spanlastics to Augment the Antifungal Activity Against Candida Albicans

pharmaceutics

Article Development and Optimization of Luliconazole Spanlastics to Augment the Antifungal Activity against Candida albicans

Nabil A. Alhakamy 1,2,3 , Mohammed W. Al-Rabia 4, Shadab Md 1,2,3 , Alaa Sirwi 5 , Selwan Saud Khayat 1, Sahar Saad AlOtaibi 1, Raghad Abkar Hakami 1, Hadeel Al Sadoun 6, Basmah Medhat Eldakhakhny 7 , Wesam H. Abdulaal 8 , Hibah M. Aldawsari 1,3 , Shaimaa M. Badr-Eldin 1,9,* and Mahmoud A. Elfaky 5

1 Department of Pharmaceutics, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] (N.A.A.); [email protected] (S.M.); [email protected] (S.S.K.); [email protected] (S.S.A.); [email protected] (R.A.H.); [email protected] (H.M.A.) 2 Advanced Drug Delivery Research Group, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia 3 Center of Excellence for Drug Research and Pharmaceutical Industries, King Abdulaziz University, Jeddah 21589, Saudi Arabia 4 Department of Medical Microbiology and Parasitology, Faculty of Medicine, King Abdulaziz Universit, Jeddah 21589, Saudi Arabia; [email protected] 5 Department of Natural Products and Alternative, Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] (A.S.); [email protected] (M.A.E.) Citation: Alhakamy, N.A.; Al-Rabia, 6 Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, King Fahd Medical M.W.; Md, S.; Sirwi, A.; Khayat, S.S.; Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] AlOtaibi, S.S.; Hakami, R.A.; Al 7 Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Sadoun, H.; Eldakhakhny, B.M.; Jeddah 21589, Saudi Arabia; [email protected] 8 Abdulaal, W.H.; et al. Development Department of Biochemistry, Faculty of Science, Cancer and Mutagenesis Unit, King Fahd Medical Research and Optimization of Luliconazole Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia; [email protected] 9 Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Spanlastics to Augment the Cairo 11562, Egypt Antifungal Activity against Candida * Correspondence: [email protected]; Tel.: +966-598281986 albicans. Pharmaceutics 2021, 13, 977. https://doi.org/10.3390/ Abstract: Luliconazole is a new topical imidazole antifungal drug for the treatment of skin infections. pharmaceutics13070977 It has low solubility and poor skin penetration which limits its therapeutic applications. In order to

Academic Editors: Maria José improve its therapeutic efficacy, spanlastics nanoformulation was developed and optimized using a Soares Mendes Giannini, combined mixture-process variable design (CMPV). The optimized formulation was converted into a Luigi Scipione, Marcia S.C. Melhem hydrogel formula to enhance skin penetration and increase the efficacy in experimental cutaneous and Ana Marisa Fusco Almeida Candida albicans infections in Swiss mice wounds. The optimized formulation was generated at percentages of Span and Tween of 48% and 52%, respectively, and a sonication time of 6.6 min. The Received: 2 May 2021 software predicted that the proposed formulation would achieve a particle size of 50 nm with a Accepted: 22 June 2021 desirability of 0.997. The entrapment of luliconazole within the spanlastics carrier showed significant Published: 28 June 2021 (p < 0.0001) antifungal efficacy in the immunocompromised Candida-infected Swiss mice without causing any irritation, when compared to the luliconazole treated groups. The microscopic obser- Publisher’s Note: MDPI stays neutral vation showed almost complete removal of the fungal colonies on the skin of the infected animals with regard to jurisdictional claims in (0.2 ± 0.05 log CFU), whereas the control animals had 0.2 ± 0.05 log CFU. Therefore, luliconazole published maps and institutional affil- spanlastics could be an effective formulation with improved topical delivery for antifungal activity iations. against C. albicans.

Keywords: luliconazole; spanlastics; particle size; Candida albicans infections; lesion score

Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and conditions of the Creative Commons Luliconazole is a new topical imidazole antifungal that belongs to the azoles group Attribution (CC BY) license (https:// with broad-spectrum antifungal activity against pathogenic fungi [1]. It is used as a creativecommons.org/licenses/by/ treatment for superﬁcial infections such as dermatophytosis and candidiasis by inhibiting 4.0/). the ability of fungi to grow and reproduce [2]. It was invented at the Research Center

Pharmaceutics 2021, 13, 977. https://doi.org/10.3390/pharmaceutics13070977 https://www.mdpi.com/journal/pharmaceutics Pharmaceutics 2021, 13, 977 2 of 11

at Nihon Nohyaku Co. Ltd. (Osaka, Japan) then approved in Japan in 2005 as a topical agent [3]. It has lately been approved by the U.S. Food and Drug Administration [4]. It has a minor aqueous solubility which lessens its dermal availability and acts as a hurdle for topical delivery [5]. The most prevalent cutaneous infections by Candida are superficial infections of skin and mucous membranes [6]. It occurs in skin folds, the genitals, cuticles, oral mucosa, or any other part of the body. The signs and symptoms are dependent on the site of infection. Cutaneous infections must be treated by drying and antifungal agents [7]. Although acquired resistance is less common than intrinsic resistance, many studies suggest that it is beginning to emerge for many antifungal drugs in some countries. For example, many studies have reported the ability of Candida albicans to develop resistance to fluconazole [8,9]. Oxman and colleagues [8] found that 19% of their Candida infections comprised either fluconazole resistant strains or strains with impaired susceptibility in their recent retrospective case-comparator study undertaken at two tertiary-care cancer centres in Boston, MA, USA. C. albicans, C. tropicalis, and C. parapsilosis, which are considered to be fluconazole sensitive, accounted for 36% of reduced susceptibility isolates and 48% of resistant isolates. Resistance identified in this species may be due to the presence of ERG11 point mutations [10], overexpression of ERG11 [11], and the overexpression of drug efflux pumps [12]. Recently, various topically applied nanocarrier systems have been developed, including lipid based nanocarriers and polymer based nanocarriers, due to their unique advantages and great versatility as compared to conventional formulations [13–17]. In one of the studies, a nanosponge system was found to confine the drug into the pores of this system that showed a good availability of drugs, enhanced skin permeability and extended retention time at the site of application [5]. As well, they found that making the drug in a gel form could present a synergistic effect of extending the retention time due to the high viscosity of this formulation. One more study showed that luliconazole-loaded nanostructured lipid carrier formulations, including lipids and span as a surfactant, had been prepared that indicated better skin penetration, increased retention time and prevented sys- temic toxicity [18]. Ksur and coworkers developed elastic lipogel and ethogel formulations of luliconazole which included sodium deoxycholate and Tween 80 as an edge activator, in addition to soya phosphatidylcholine as phospholipid and ethanol were found to be more effective against dermatophytes in comparison with the approved formulation [19]. The spanlastics method, which is a new drug delivery system that would directly target the infected cells with minor side effects, works by encapsulating the drug in a vesicle made by a nonionic surfactant that acts as a carrier of the drug to deliver it to the target site. In addition, this nonionic surfactant has good compatibility with biological systems and represents low toxicity. The elastic vesicles refer to the existence of edge activators in their structure [20]. Therefore, the objectives of the study were to prepare and optimize luliconazole spanlastics as potential carriers for topical delivery, using a combined mixture-process variable design (CMPV). Luliconazole spanlastics were prepared which were later converted into hydrogels. The optimized luliconazole spanlastics were evaluated for the experimental cutaneous C. albicans infections in Swiss mice wounds.

2. Materials and Methods 2.1. Materials Luliconazole was procured as a gift sample from Jamjoom Pharmaceutical, Jeddah, Saudi Arabia. Span 20 and Tween 20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were of analytical grade.

2.2. Experimental Design Combined mixture-process variable design (CMPV) was employed for the formulation and optimization of Luliconazole spanlastics. The design was chosen based on its ability to evaluate how the responses are affected by the mixture elements and the process variables simultaneously. In this study, the mixture was composed of two mixture components, Pharmaceutics 2021, 13, 977 3 of 11

namely Span 20 (A) and Tween 20 (B); both were used in percentages ranging from 10–90% so that the total mixture added up to 100%. Sonication time (C) was investigated as process variable (PV) in the range of 0–10 min. The remaining process parameters were kept unchanged in all experimental runs. Particle size (PS, nm) (Y) was considered as a response. The investigated variables with their investigated ranges, in addition to the response and the constraints of the optimization process are compiled in Table1. Design Expert software (Version 11.0, Stat- Ease Inc., Minneapolis, MN, USA) was applied for the design points generation and statistical analysis of the data obtained. The selection of the design points was based on the D-optimal design where the total runs were 17 and included required model points, lack of ﬁt points, replicate points and additional center points, Table2.

Table 1. Ranges of mixture components and process variables and the desired constraints of the response variables for the CMPV design.

Mixture Components Lower Level Upper Level A: Span 20 percentage 10 90 B: Tween 20 percentage 10 90 Process Variable C: Sonication time (min) 0 10 Responses Desirability Constraints Y: Particle size (PS, nm) Minimize Abbreviations: CMPV, combined mixture process variable.

Table 2. The variables’ levels and the observed responses of luliconazole spanlastics runs.

Mixture Components Process Variables Run No. Particle Size (nm) ABC 1 90 10 10 307.6 2 10 90 10 701.9 3 50 50 0 198.0 4 10 90 5 256.2 5 30 70 2.5 166.1 6 30 70 7.5 330.7 7 90 10 0 1204.0 8 10 90 0 4174.0 9 70 30 7.5 324.1 10 90 10 0 2130.0 11 70 30 2.5 327.5 12 90 10 10 771.5 13 10 90 10 367.6 14 30 70 0 5891.0 15 90 10 5 218.2 16 50 50 10 60.8 17 50 50 5 143.6 Abbreviations: A, Span percentage; B, Tween percentage; C, sonication time (min).

2.3. Preparation of Luliconazole Spanlastics Seventeen formulations of spanlastics named as F1–F17 were developed using different percentages of Span 20 and Tween 20 as per the CMPV design using a previously reported method [21]. The drug was kept constant at 20 mg in each formulation. The drug and specified amount of Span were dissolved in 5 mL of ethanol, while Tween as an edge activator was dissolved in 10 mL of distilled water at a temperature of 65 ◦C. The alcoholic solution was injected dropwise into the aqueous solution that was stirred on a magnetic stirrer at a speed of 1000 rpm. The final mixture was kept on the stirrer for an additional 30 min at room temperature. The formed dispersion was then subjected to ultrasonication for the time specified in the design. Pharmaceutics 2021, 13, 977 4 of 11

2.4. Particle Size Measurement For particle size determination, the dispersions were diluted appropriately with distilled water and then particle size was measured using Zetasizer (Malvern Panalytical Ltd., Malvern, UK) particle size cells. Each sample was measured in triplicate.

2.5. Optimization of Luliconazole Spanlastics The optimized levels for the mixture components and the sonication time were predicted by applying the numerical optimization technique based on desirability approach.

2.6. TEM Investigation of the Optimized Luliconazole Splanlastics Formula The optimized formula dispersion (one drop) was spread on a carbon grid and then phosphotungstic acid was utilized for staining. After that, the sample was dried, and then investigated by TEM (JEM-1011: JEOL, Tokyo, Japan).

2.7. Assessment of Antifungal Activity of the Optimized Luliconazole Spanlastics 2.7.1. In Vitro Antifungal Susceptibility Testing Determination of MIC of Luliconazle and Luliconazole spanlastics against C. albicans isolates were conducted in accordance with Clinical Laboratory Standards Institute guide- lines using broth microdilution method. Isolates were tested in triplicate and the MIC mode was determined.

2.7.2. Animals Male adult animals (Swiss mice) weighing 25–30 g were obtained from the animal house of the Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia. The in vivo study protocol was approved by the Animal Ethics Committee of the Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia, ethical approval no. PH-127- 41 (2021), in adherence with the Declaration of Helsinki, the Guiding Principle in Care and Use of Animals (DHEW production NIH 80-23) and the Standards of Laboratory Animal Care (NIH distribution #85-23, reconsidered in 1985). Animals were adapted for at least 2 weeks in naturally controlled enclosures (20 ± 1 ◦C and a 12/12-h dark/light cycle) and were fed pelleted food and tap water ad libitum.

2.7.3. Candida and Culture Conditions C. albicans (ATCC 90028) was kept on Sabouraud’s dextrose agar (SDA) plate for 48 h at 35 ◦C. Colonies were suspended in 5 mL 0.85% sterile phosphate buffer saline (PBS). The ﬁnal concentration was adjusted to 2 × 107 CFU/mL.

2.7.4. Animal Preparation and Cutaneous Infections Twenty adult Swiss mice were randomly divided into four groups (n = 5/group). Mice were intraperitoneally injected by cyclophosphamide (100 mg/kg/day) for three days prior to fungal infection to induce immunocompromised animals [22]. Induction of cutaneous candidiasis model was made following Maebashi et al. [2] with some modiﬁcation. The backs of the mice were shaved with an electronic shaver (area 2 × 2 cm). After 48 h, the skin was moderately abraded with sandpaper. One hundred microliters of Candida cell suspension (2 × 107 cells) were gently applied by cotton-tipped swab onto the skin.

2.7.5. Treatment of the Infection Dosing was giving once a day for ﬁve consecutive days, starting 24 h after Candida infection. Group 1 was negative control with no infection and no treatment (control group), group 2 was positive control receiving infection with no treatment (untreated group), group 3 applied topically with Luliconazole 25 mg/kg/day (Luli group), and group 4 applied topically with equivalent dose of Luliconazole spanlastics 25 mg/kg/day (Luli-span group). Pharmaceutics 2021, 13, 977 5 of 11

2.7.6. Evaluation of Infection Macroscopic evaluation of infection was measured by a score of erythema from 0 to 4 as described (2): 0, not erythematous (normal); 1, slightly erythematous; 2, moderately erythematous; 3, spreading erythematous; and 4, severely erythematous. For microbiological evaluation of infection, mice were sacriﬁced 24 h following the last dose and 1 × 1 cm of skin from the infected sites was excised. The infected skin samples were washed with sterile PBS and plated onto SDA plates then incubated for 48 h at 37 ± 1 ◦C, and CFU values of C. albicans were recorded. Data were presented as mean ± standard error and were statistically analyzed using one-way ANOVA (SPSS, Inc., Chicago, IL, USA) to measure the differences between the parameters. The conﬁdence level using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA) was set at * p < 0.01 and ** p < 0.001.

3. Results and Discussion 3.1. Particle Size Results and Model Fit Statistical Analysis The variables’ levels for the luliconazole spanlastics runs and the measured particle size for each run are displayed in Table2. The mean particle size exhibited a marked variation ranging from 60.8 to 5891 nm. The ﬁt statistics analysis was carried out to produce a CMPV polynomial model illustrating the relation between the particle size and the studied MCs and PV. The statistical software suggested the quadratic × cubic (Q × C) based on the highest adjusted R2 (0.9436). In addition, adequate precision of 18.05 (greater than 4) indicated appropriate signal to noise ratio [23]. Accordingly, the suggested model could be applied to navigate the experimental space. The polynomial equation that describes the particle size in terms of L-Pseudo components of the mixture and coded factor for the sonication time was developed by the software as follows:

Y = 222.28 A + 260.82 B − 281.94 AB − 553.32 AC − 1804.21 BC + 4940.31 ABC + 880.84 AC2 + 2088.93 BC2 + 19,106.27 AB(A − B) − 6088.71 ABC2 − 4231.70 ABC(A − B) − 71,146.97 ABC2(A − B)

The equation in terms of coded factors can be utilized for predicting the response for given levels of each factor. The high levels of the mixture components and process factors were coded as +1, the low levels of the mixture components were coded as 0, and the low levels of the process factors were coded as −1. The coded equation is advantageous for finding the relative influence of the factors by comparing the corresponding coefficients. The presence of significant MPV coefficients in the equations highlights the usefulness of employing the CMPV design as it reveals the interaction between the MCs and the PV; such interaction could never be detected using a traditional one factor at a time approach or even experimental designs done individually on MCs and PVs [24,25].

3.2. Diagnostics for the Validity of the Quadratic Model A diagnostic plot of externally studentized residuals versus run for particle size was generated to establish the goodness of ﬁt of the quadratic model. Figure1 displays randomly distributed points showing that no lurking variable could inﬂuence the measured response [15,26].

3.3. Influence of Variables on Particle Size (PS, Y1) Reducing the particle size of the vesicular systems to the nano-range could markedly contribute to promoting the absorption of active ingredients and/or improving their biological performance [27]. The Q × C model was significant for the particle size data (p = 0.0011). The computed F-values of 25.23 confirmed the significance of the model; there was only a chance of 0.11% that such a value could be this large owing to noise. Lack of fit F-values of 0.37 showed a nonsignificant lack of fit in relation to the pure error indicating fitting of the data to the model. According to the computed p-values, the terms AC, BC, Pharmaceutics 2021, 13, x 6 of 12

The equation in terms of coded factors can be utilized for predicting the response for given levels of each factor. The high levels of the mixture components and process factors were coded as +1, the low levels of the mixture components were coded as 0, and the low levels of the process factors were coded as −1. The coded equation is advantageous for finding the relative influence of the factors by comparing the corresponding coefficients. The presence of significant MPV coefficients in the equations highlights the usefulness of employing the CMPV design as it reveals the interaction between the MCs and the PV; such interaction could never be detected using a traditional one factor at a time approach or even experimental designs done individually on MCs and PVs [24,25].

Pharmaceutics 2021, 13, 977 3.2. Diagnostics for the Validity of the Quadratic Model 6 of 11 A diagnostic plot of externally studentized residuals versus run for particle size was generated to establish the goodness of fit of the quadratic model. Figure 1 displays ran- domlyABC, BC distributed2, AB(A − pointsB), ABC showing2, ABC2 that(A − noB) lurking were signiﬁcant variable (couldp < 0.05) influence on the measuredthe measured data. responseThe three-dimensional [15,26]. mixture-process plot for the particle size are presented in Figure2.

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FigureFigure 1. 1. ExternallyExternally studentized studentized residuals residuals vs. vs. run run numb numberer for the for quadratic the quadratic × cubic× cubicmodel model for lu- for liconazoleluliconazole spanlastics spanlastics particle particle size. size.

3.3. Influence of Variables on Particle Size (PS, Y1) Reducing the particle size of the vesicular systems to the nano-range could markedly contribute to promoting the absorption of active ingredients and/or improving their biological performance [27]. The Q × C model was significant for the particle size data (p = 0.0011). The computed F-values of 25.23 confirmed the significance of the model; there was only a chance of 0.11% that such a value could be this large owing to noise. Lack of fit F-values of 0.37 showed a nonsignificant lack of fit in relation to the pure error indicating fitting of the data to the model. According to the computed p-values, the terms AC, BC, ABC, BC2, AB(A − B), ABC2, ABC2(A − B) were significant (p < 0.05) on the measured data. The three-dimensional mixture-process plot for the particle size are presented in Figure 2.

FigureFigure 2. Effect 2. Effect of the ofbinary the binarymixture mixture components components (I) and the (I) sonication and the sonication time (II) at time the mid-values (II) at the mid- of thevalues other ofvariables; the other Three-dimensional variables; Three-dimensional mixture−process mixture plot− (III)process for the plot particle (III) for size the of particle lulicona- size of zole luliconazolespanlastics. spanlastics.

3.4. 3.4.Optimization Optimization Using Using a Numerical a Numerical Approach Approach The Theaim aimof optimization of optimization in the in the formulation formulation field ﬁeld is isto topredict predict the the values values of of the the varia- variables blesthat that couldcould yieldyield aa product with the desire desiredd characteristics. characteristics. In In our our study, study, the the optimi- optimiza- zationtion process process aims aims at at obtaining obtaining Luliconazole Luliconazole spanlastics with minimizedminimized particleparticle size.size. The The numerical optimization technique was adopted to achieve this aim. The optimized formulation was generated at percentages of Span and Tween of 48% and 52%, respectively, and a sonication time of 6.6 min. The software predicted that the proposed formulation could achieve a globule size of 50 nm with a desirability of 0.997.

3.5. Transmission Electron Microscope Investigation of the Optimized Luliconazole Spanlastics Transmission electron microscope (TEM) photographs of the optimized formula showed rounded structures which revealed some aggregations that could be attributed to the process of drying during sample preparation (Figure 3).

Figure 3. Transmission electron microscope photograph of optimized luliconazole spanlastics.

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Figure 2. Effect of the binary mixture components (I) and the sonication time (II) at the mid-values of the other variables; Three-dimensional mixture−process plot (III) for the particle size of luliconazole spanlastics.

3.4. Optimization Using a Numerical Approach Pharmaceutics 2021, 13, 977 7 of 11 The aim of optimization in the formulation field is to predict the values of the variables that could yield a product with the desired characteristics. In our study, the optimization process aims at obtaining Luliconazole spanlastics with minimized particle size. numericalThe numerical optimization optimization technique technique was adopted was adop toted achieve to achieve this aim. this The aim. optimized The optimized formu- lationformulation was generated was generated at percentages at percentages of Span of and Span Tween and of Tween 48% and of 52%,48% and respectively, 52%, respec- and atively, sonication and atime sonication of 6.6 min.time Theof 6.6 software min. The predicted software that predicted the proposed that the formulation proposed formu- could achievelation could a globule achieve size a ofglobule 50 nm size with of a 50 desirability nm with a of desirability 0.997. of 0.997.

3.5.3.5. Transmission ElectronElectron MicroscopeMicroscope InvestigationInvestigationof ofthe theOptimized OptimizedLuliconazole Luliconazole Spanlastics Spanlastics TransmissionTransmission electronelectron microscopemicroscope (TEM)(TEM) photographsphotographs ofof thethe optimizedoptimized formulaformula showedshowed roundedrounded structuresstructures whichwhich revealedrevealed somesome aggregationsaggregations thatthat couldcould bebe attributedattributed toto thethe processprocess ofof dryingdrying duringduring samplesample preparationpreparation (Figure(Figure3 ).3).

FigureFigure 3.3. TransmissionTransmission electronelectron microscopemicroscope photographphotograph ofof optimizedoptimized luliconazoleluliconazole spanlastics. spanlastics.

3.6. In Vitro Antifungal Susceptibility Testing An in vitro antifungal susceptibility study using broth microdilution was carried out where the minimum inhibitory concentrations (MIC) of the luliconazole spanlastics were compared with those of luliconazole alone against C. albicans (ATCC 90028). Results revealed no differences in the susceptibility patterns between the tested components.

3.7. In Vivo Study Being the most opportunistic pathogen for human infection, C. albicans are known to invade superficially and simultaneously spread fungal infection. Furthermore, the growing emergence of developing resistance by the fungal species and the unfavorable outcomes of the conventional treatments urges novel deliveries [28]. Five days consecutive cutaneous treatment of the immunocompromised animals with Candida infection showed promising results as depicted in Figures4 and5. The animals in Group 1 did not show any signs of infection as clearly shown in Figure4A,B, whereas the average lesion score of the infected animals was demonstrated to decrease with the days of treatments. As there was no treatment provided to the infected animals in Group 2, the rate of subsiding of the infection in those animals is much less. On the other hand, treatment of the infected animals with luliconazole was found to decrease the average lesion score of the animals in Group 3 (Figure4A,B). Our findings on the obtained antifungal efficacy of luliconazole (25 mg/kg/day) showed agreement with the existing literature where the control of bacterial infection using luliconazole was equivalent [29]. Furthermore, treatment of luliconazole spanlastics to the animals in group 4 showed outstanding control of infections as depicted by the significant (p < 0.0001) reduction in the lesion score with significant reduction in the erythema score when compared to the results obtained in Group 3, treated with luliconazole (Figures3 and4). This significant recovery of the animals was clearly demonstrated in Figure4, where the gradual recovery of the experimental animals administered with luliconazole spanlastics is clearly displayed. This superior efficacy of the drug (luliconazole) using a spanlastics platform over the plain luliconazole treatment might be explained by the preeminence in topical delivery of luliconazole-loaded surfactant-based elastic nanovesicles [30]. Pharmaceutics 2021, 13, x 8 of 12

3.6. In vitro Antifungal Susceptibility Testing An in vitro antifungal susceptibility study using broth microdilution was carried out where the minimum inhibitory concentrations (MIC) of the luliconazole spanlastics were compared with those of luliconazole alone against C. albicans (ATCC 90028). Results revealed no differences in the susceptibility patterns between the tested components.

3.7. In Vivo Study Being the most opportunistic pathogen for human infection, C. albicans are known to invade superficially and simultaneously spread fungal infection. Furthermore, the growing emergence of developing resistance by the fungal species and the unfavorable outcomes of the conventional treatments urges novel deliveries [28]. Five days consecutive Pharmaceutics 2021, 13, 977 8 of 11 cutaneous treatment of the immunocompromised animals with Candida infection showed promising results as depicted in Figures 4 and 5.

Figure 4. EstablishmentFigure 4. Establishment of infection of expressed infection expressed as lesion score as lesion after score starting after treatment starting treatment in cutaneous in cutaneous candidiasis mouse model. Micecandidiasis (n = 5/group) mouse were model. applied Mice topically (n = 5/group) with luliconazolewere applied 25 topically mg/kg/day with luliconazole as Luli group 25 and equivalent dose of luliconazolemg/kg/day spanlastics as Luli (Luli-span group and group) equivalent for 5 days. dose (ofA )luliconazole The score of spanlastics erythema (Luli-span were measured group) on for days 5 1, 3 and 5. (B) Averagedays. lesion (A) score The inscoreC. albicansof erythemainduced were cutaneous measured candidiasison days 1, 3 in and mice 5. (B treated) Average with lesion different score formulations in C. and albicans induced cutaneous candidiasis in mice treated with different formulations and compared compared with control and untreated group on the 5th day. Data were presented as mean ± SD. @ significant Luli span vs. with control and untreated group on the 5th day. Data were presented as mean ± SD. @ significant untreated (p < 0.0001). * Significant Luli span vs. Luli group (p < 0.0001) determined by student t test. Luli span vs. untreated (p < 0.0001). * Significant Luli span vs. Luli group (p < 0.0001) determined by student t test. Furthermore, the inoculation of C. albicans to the abraded skin of the animals showed white patches composed of microscopic colonies when observed under microscope. There were no colonies observed in the control group, whereas the number of colonies in the untreated animals after five days of inoculation was recorded as 14 ± 2.2 log CFU at the infected site (Table3). This number was found to be decreased significantly ( p < 0.0001) in the luliconazole treated animals when compared to the untreated animal groups. Moreover, the log colonies in the animals of the luliconazole spanlastics treatment group was found to 0.2 ± 0.05, which is statistically significant (p < 0.0001) when compared to the results of luliconazole treatment group. Thus, the microscopic findings of the animals treated with the spanlastics delivery is comparable to the macroscopic findings of the animal skin. PharmaceuticsPharmaceutics 20212021,, 1313,, x 977 9 9of of 12 11

FigureFigure 5. 5. MacroscopicMacroscopic evaluation evaluation of of infection infection was was measured measured by by a score a score of oferythema erythema in inmice mice of ofall all threethree groups groups at at 1, 1, 3 3 and and 5 5 days. days. On On the the 1st 1st day day after after induction induction of of C.C. albicans albicans cutaneouscutaneous candidiasis, candidiasis, obviousobvious infection infection developed developed in in all all groups. groups. On On 3rd 3rd day day small small recovery recovery was was observed observed in in lulicona- luliconazole zole(Luli (Luli group) group) and and luliconazole luliconazole spanlastics spanlastics (Luli-span (Luli-span group). group). On On 5th 5th day day signiﬁcant significant recovery recovery was wasobserved observed in Luli-span in Luli-span group. group.

TableThe 3. Colony-forming animals in Group units of1 didC. albicans not showon the any skin sign of mices of after infection treatment as clearly with different shown formulations. in Figure 4A,B, whereas the average lesion score of the infected animals was demonstrated to de- Treatment Log CFU Infected creaseSample with No. the days of treatments. As there wasNumber no treatment of Animals provided to the infected (groups) Site animals in Group 2, the rate of subsiding of the infection in those animals is much less. On the other1 hand, treatment Control of the infected animals with 5 luliconazole was found 0 to decrease the2 average lesion score Untreated of the animals in Group 53 (Figure 4A,B). Our 14 ±findings2.2 on 3 Luliconazole 5 3.1 ± 0.2 the obtained antifungal efficacy of luliconazole (25 mg/kg/day) showed agreement with 4 Luliconazole spanlastics 5 0.2 ± 0.05 * the existing literature where the control of bacterial infection using luliconazole was Values represent mean ± SD. * Signiﬁcant reduction in fungal count of Luliconazole spanlastics versus Lulicona- equivalentzole treatment [29]. (* p

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Author Contributions: Conceptualization, N.A.A. and S.M.B.-E.; methodology, S.M., R.A.H., A.S. and H.A.S.; software, S.M.B-E.; validation, M.W.A.-R., H.M.A.; formal analysis, S.S.K., S.S.A.; investigation, M.A.E. and A.S.; resources, N.A.A. and W.H.A.; data curation, B.M.E. and H.M.A.; writing—original draft preparation, S.M., N.A.A.; writing—review and editing, S.M. and S.M.B.-E.; visualization, W.H.A., and H.M.A.; supervision, M.W.A.-R.; project administration, N.A.A.; funding acquisition, N.A.A. All authors have read and agreed to the published version of the manuscript. Funding: This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. (RG-16-166-41). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are available within the manuscript. Acknowledgments: This project was funded by the Deanship of Scientific Research (DSR) at King Abdulaziz University, Jeddah, under grant no. RG-16-166-41. The authors, therefore, acknowledge with thanks DSR for technical and financial support. Conflicts of Interest: The authors declare no conflict of interest.

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