pharmaceuticals Article Development of a Topical Amphotericin B and Bursera graveolens Essential Oil-Loaded Gel for the Treatment of Dermal Candidiasis Lupe Carolina Espinoza 1,2 , Lilian Sosa 3 , Paulo C. Granda 2,4 , Nuria Bozal 5 , Natalia Díaz-Garrido 6,7 , Brenda Chulca-Torres 1 and Ana Cristina Calpena 2,4, * 1 Departamento de Química, Universidad Técnica Particular de Loja, Loja 1101608, Ecuador; lcespinoza@utpl.edu.ec (L.C.E.); bachulca@utpl.edu.ec (B.C.-T.) 2 Institute of Nanoscience and Nanotechnology (IN2UB), University of Barcelona, 08028 Barcelona, Spain; paulogranda92@gmail.com 3 Faculty of Chemical Sciences and Pharmacy, National Autonomous University of Honduras (UNAH), Tegucigalpa 11101, Honduras; lilian.sosa@unah.edu.hn 4 Department of Pharmacy, Pharmaceutical Technology and Physical Chemistry, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain 5 Department of Biology, Healthcare and the Environment, School of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain; nuriabozaldefebrer@ub.edu 6 Department of Biochemistry and Physiology, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain; natalia.diaz.garrido@gmail.com 7 Institute of Biomedicine of the University of Barcelona-Sant Joan de Déu Research Institute (IBUB-IRSJD),   08028 Barcelona, Spain * Correspondence: anacalpena@ub.edu Citation: Espinoza, L.C.; Sosa, L.; Granda, P.C.; Bozal, N.; Díaz-Garrido, Abstract: The higher molecular weight and low solubility of amphotericin B (AmB) hinders its N.; Chulca-Torres, B.; Calpena, A.C. topical administration. The aim of this study was to incorporate Bursera graveolens essential oil Development of a Topical into an AmB topical gel (AmB + BGEO gel) in order to promote the diffusion of the drug through Amphotericin B and Bursera the skin in the treatment of cutaneous candidiasis. AmB + BGEO gel formulation was determined graveolens Essential Oil-Loaded Gel for the Treatment of Dermal using a factorial experiment. Physical and chemical parameters, stability, in vitro release profile Candidiasis. Pharmaceuticals 2021, 14, and ex vivo permeation in human skin were evaluated. In vitro antimicrobial activity was studied 1033. https://doi.org/10.3390/ using strains of C. albicans, C. glabrata and C. parapsilosis. The tolerability was evaluated using ph14101033 in vitro and in vivo models. AmB + BGEO gel presented appropriate characteristics for topical administration, including pH of 5.85, pseudoplastic behavior, optimal extensibility, as well as high Academic Editor: Noelia Duarte stability and acceptable tolerability. In vitro release studies showed that the formulation releases the drug following a Boltzmann sigmoidal model. Finally, AmB + BGEO gel exhibited higher amount Received: 10 September 2021 of drug retained inside the skin and lower Minimum Inhibitory Concentration than a formulation Accepted: 4 October 2021 sans essential oil. Therefore, these results suggest that the incorporation of B. graveolens essential oil Published: 12 October 2021 in the formulation could be used as strategy to promote a local effect in the treatment of cutaneous candidiasis. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Keywords: amphotericin B; candidiasis; gel; essential oil; Bursera graveolens; topical treatment published maps and institutional affil- iations. 1. Introduction Copyright: © 2021 by the authors. Cutaneous candidiasis is a superficial mycosis caused by proliferation in the skin by Licensee MDPI, Basel, Switzerland. fungal organisms belonging to the genus Candida [1]. C. albicans is responsible for approx- This article is an open access article imately 70% of skin infections associated with Candida spp. [2,3]. This type of mycosis distributed under the terms and mainly affects intertriginous areas and produces dryness, erythema, edema, erosions and conditions of the Creative Commons pustules [4]. Topical treatments for cutaneous candidiasis include the application of azoles Attribution (CC BY) license (https:// and polyenes such as clotrimazole, miconazole, fluconazole, itraconazole and nystatin [5–7]. creativecommons.org/licenses/by/ However, resistance mechanisms both intrinsic and acquired to these antifungal drugs 4.0/). have been reported. Intrinsic resistance is highly stable and is predictive of therapeutic Pharmaceuticals 2021, 14, 1033. https://doi.org/10.3390/ph14101033 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, 1033 2 of 15 failure whereas acquired resistance can be either stable or transient and is developed due to long exposure to antifungals [8–10]. Although polyene resistance is still uncommon compared to resistance to other antifungals, some mechanisms of resistance have been proposed, including: (1) alterations in the sterol composition of the fungal cell membrane due to several mutations in genes (ERG) of the ergosterol biosynthesis pathway, (2) oxida- tive stress, and (3) alterations of the fungal cell wall characterized by an increased glucan production [11]. AmB is a polyene macrolide antibiotic produced by Streptomyces nodosus that acts by binding with the sterols (ergosterol) present in the cell membrane of susceptible fungi, resulting in the creation of transmembrane channels that induce ion permeability, loss of protons and monovalent cations, and consequently depolarization and concentration- dependent cell killing [12,13]. AmB has been clinically used to treat Candida, Cryptoccocus and Aspergillus via intravenous infusion [14,15]. However, its systemic adverse effects are well-known, including symptoms from acute toxicity such as nausea, vomiting, rigors, fever, hypertension or hypotension, and hypoxia as well as chronic adverse effects such as nephrotoxicity [16]. A topically administered AmB formulation can bypass these disad- vantages from invasive administration techniques and thus permit treatment of localized infections in skin or mucous membranes [17]. However, the higher molecular weight (926 Da) and low solubility in water of AmB could limit its passage through the skin [18]. These complications could be circumvented by incorporating excipients with permeation- enhancing properties that promote the penetration of drugs through the stratum corneum (SC) and its distribution to the epidermis and dermis [19]. Examples of commonly studied permeation enhancers for dermal drug delivery include azone, pyrrolidones, sulphoxides, fatty acids, and surfactants as well as essential oils and their components [20]. Ideally, these compounds induce a temporary and reversible reduction in the barrier function of the SC in order to facilitate the drug permeation across the skin [21,22]. Essential oils are natural products extracted from aromatic plant materials and contain complex mixtures of several volatile compounds such as terpenes, terpenoids and phenyl- propanoids [22]. Essential oils are widely used in pharmaceutical, cosmetic, agricultural and food industries in several applications including improvement of drug permeation in addition to antibacterial, antifungal, antioxidant and anti-inflammatory activities [23]. As permeation enhancers, the active compounds derived from essential oils increase drug diffusion by changes in the SC structure and interaction with intercellular lipids in the SC [22,23]. In particular, the Bursera graveolens essential oil has been reported to inhibit the growth of breast tumor cells and amastigotes of Leishmania amazonensis while also having antioxidant, antifungal and anticarcinogenic properties [24,25]. B. graveolens is a deciduous tree found from western Mexico to northwestern Peru. Its wood exhibits a characteristic sweet, spicy and balsamic aroma that has traditionally been used as in- cense [26]. It is widely referred to as “palo santo” by local populations, which in Spanish means “holy wood” [27]. Essential oil obtained from the fruit of B. graveolens primarily contains D-limonene (49.89%), α-phellandrene (37.64%) and menthofuran (6.08%) [25,27]. The purpose of this study was to develop and characterize a topical gel of AmB and B. graveolens essential oil (AmB + BGEO gel) as strategy to promote drug permeation through the SC and its retention in human skin in order to achieve a local effect in the treatment of cutaneous candidiasis. 2. Results 2.1. Solubility Studies The AmB solubility in different components are shown in Figure 1. DMSO (solubility 3.82 ± 0.05 mg/mL) exhibited greater ability to solubilize the drug and was, therefore, used as solvent in the formulation. Propylene glycol 400 (solubility 1.09 ± 0.04 mg/mL) and glycerin (solubility 1.02 ± 0.04 mg/mL) were evaluated by a 23 factorial experiment in order to determine the most appropriate cosolvent for the formulation. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 15 Pharmaceuticals 2021, 14, 1033 3 of 15 glycerin (solubility 1.02 ± 0.04 mg/mL) were evaluated by a 23 factorial experiment in order to determine the most appropriate cosolvent for the formulation. DMSO Glycerin Propylene glycol 400 Castor oil Labrafac™ lipophile WL 1349 Plurol® oleique CC497 Lauroglycol™ 90 0 1 2 3 4 5 Solubility (mg/mL) Figure Figure1.1.Amphotericin AmphotericinBB(AmB) (AmB)solubility solubilityinindifferent differentexcipients excipients(n(n==3). 3). 2.2.Design 2.2. Designand andPreparation PreparationofofAmB+BGEO AmB + BGEO GelGel Formulation Formulation Table 1 shows the physical and chemical characterizationof Table 1 shows the physical and chemical characterization ofthe theeight eightformulations formulations obtainedby 3 by223 factorial factorialexperiment. experiment.AfterAfter6060days daysofofstorage storageatat30 ◦ 30°C,C,formulations formulations1,1,4,4,66 obtained and 8 showed precipitates or formed lumps, whereas formulations 2 and 5 5were and 8 showed precipitates or formed lumps, whereas formulations 2 and werehomoge- homoge- neous but showed significant changes in pH. These signs of instability were neous but showed significant changes in pH. These signs of instability were accompanied accompanied bya adecrease by decreaseinindrug drug content. content. Finally, Finally, whenwhen comparing comparing formulations formulations 3 and 3 and 7, it 7, wasit ob- was observed that formulation 3 was more stable, especially with regard to served that formulation 3 was more stable, especially with regard to drug content, anddrug content, and was therefore selected as the final formulation for the AmB was therefore selected as the final formulation for the AmB+BGEO gel. + BGEO gel. Table 1. Physical and chemical characterization of the 8 formulations obtained by 23 factorial experiment after one and Table 1. Physical and chemical characterization of the 8 formulations obtained by 23 factorial experiment after one and 60 60 days days of preparation of preparation andand storage storage 30 ◦ C. at 30at°C. 11 Day Day 60 Days 60 Days Formulation Formulation Appearance Appearance pH pH Drug Content(%) Drug Content (%) Appearance Appearance pH pH Drug DrugContent Content(%)(%) F1 F1 Homogeneous Homogeneous 4.80 ± 0.03 4.80 ± 0.03 99.68 ± 0.16 99.68 ± 0.16 Precipitates Precipitates 4.12 ± 0.09 4.12 ± 0.09 96.78 ± 1.67 96.78 ± 1.67 F2 F2 Homogeneous Homogeneous 6.76 ± 6.76 0.05 ± 0.05 99.73±± 0.12 99.73 0.12 Homogeneous Homogeneous 4.98 4.98 ± 0.07 ± 0.07 98.25±± 98.25 0.82 0.82 F3 F3 Homogeneous 5.85 ± Homogeneous 5.85 ± 0.09 0.09 99.65 ± 0.08 99.65 ± 0.08 Homogeneous 5.57 Homogeneous 5.57 ± 0.04 ± 0.04 99.18 ± 0.43 99.18 ± 0.43 F4 Homogeneous 6.81 ± 0.07 99.66 ± 0.10 Lumps 6.02 ± 0.10 98.86 ± 1.35 F4 F5 Homogeneous Homogeneous 6.81 ± 5.28 ± 0.07 0.01 99.66±± 0.09 99.52 0.10 Lumps Homogeneous 6.02 ± 0.10 4.16 ± 0.15 98.86 98.16±±1.35 0.98 F5 F6 Homogeneous Homogeneous 5.28 ± 7.12 ± 0.01 0.08 99.52±± 0.13 99.59 0.09 Homogeneous Precipitates 4.16 ± 0.15 6.15 ± 0.09 98.16 97.63±±0.98 1.82 F6 F7 Homogeneous Homogeneous 7.12 ± 5.92 ± 0.08 0.04 99.59±± 0.09 99.81 0.13 Precipitates Homogeneous 5.05 ± 6.15 ± 0.09 0.07 98.83±± 97.63 1.82 0.66 F8 F7 Homogeneous 6.70 ± Homogeneous 5.92 ± 0.04 0.06 99.65 ± 0.11 99.81 ± 0.09 Precipitates 5.72 Homogeneous 5.05 ± 0.07 ± 0.25 97.15 ± 1.68 98.83 ± 0.66 F8 Homogeneous 6.70 ± 0.06 99.65 ± 0.11 Precipitates 5.72 ± 0.25 97.15 ± 1.68 AmB + BGEO gel (0.1%) was prepared by dissolving AmB in 5% DMSO, 20% Propy- AmB+BGEO gel (0.1%) was prepared by dissolving AmB in 5% DMSO, 20% Propyl- lene glycol 400, and 2% B. graveolens essential oil. This mixture was then incorporated into ene glycol 400, and 2% B. graveolens essential oil. This mixture was then incorporated into a gel base composed of 3% CMC, 0.20% citric acid, 0.02% sodium benzoate, and 69.68% a gel base composed of 3% CMC, 0.20% citric acid, 0.02% sodium benzoate, and 69.68% water (Table 2). water (Table 2). Table 2. Final formulation of AmB + BGEO gel. Table 2. Final formulation of AmB+BGEO gel. Component (%) Component (%) Amphotericin B (AmB)B (AmB) Amphotericin 0.1 0.1 B. graveolens essential oil (BGEO) 2 B. graveolens essential Carboxymethylcelullose oil (BGEO) (CMC) 3 2 Carboxymethylcelullose Propylene glycol 400 (CMC) 20 3 SodiumPropylene benzoateglycol 400 0.02 20 Citric acid benzoate Sodium 0.2 0.02 Dimethylsulfoxyde (DMSO) 5 Water Citric acid 69.68 0.2 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 15 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 15 Pharmaceuticals 2021, 14, 1033 Dimethylsulfoxyde (DMSO) 5 4 of 15 Water Dimethylsulfoxyde (DMSO) 5 69.68 Water 69.68 2.3. Characterization of AmB+BGEO Gel 2.3. Characterization of AmB + BGEO Gel 2.3. Characterization AmB+BGEO gel of AmB+BGEO Gel showed a homogeneous appearance without signs of lumps or pre- AmB + BGEO gel showed a homogeneous appearance without signs of lumps or AmB+BGEO cipitates while also gelexhibiting showed a homogeneous appearance a slightly yellowish colorwithout signs of lumps and a pleasant or pre-char- citrus scent precipitates cipitates whilewhile also exhibiting also exhibiting a slightly yellowish color and a pleasant citrus char-scent acteristic of essential oil. Theadrug slightly yellowish content in thecolor and formulationa pleasant citrus was of 99.65scent ± 0.08% and characteristic acteristic of of essential essential oil. oil. The The drug drug content content in in the the formulation formulation was was of of 99.6599.65 ± ± 0.08%0.08% and and with a pH value of 5.85 ± 0.09, which is biocompatible with the natural acidity of the skin. with a pH value with Rheological of 5.85 a pH value ofstudies ± 0.09, 5.85 ± 0.09, which which is biocompatible with the natural acidity of the skin. (Figure 2) is biocompatible confirmed with the natural pseudoplastic acidity behavior of the of the skin. formulation Rheological Rheological studies(Figure studies (Figure2)2)confirmed confirmed pseudoplastic pseudoplastic behavior behavior of of the the formulation formulation (Cross model r2 = 0.999) with a mean viscosity at 50 − 2 s of 5036 ± 45.71 mPa·s. A hysteresis −1 (Cross (Cross model model rr2 ==0.999) 0.999)withwithaamean viscosityatat5050s−1s of1 5036 meanviscosity of 5036 ± 45.71 ± 45.71 mPaA·s.hysteresis mPa·s. A hysteresis loop with an area of 4269 Pa/s was observed. loop with an loop with anarea areaof of4269 4269Pa/sPa/s was was observed. observed. Figure 2. 2. Rheological Rheologicalbehavior behaviorofofAmB+BGEO AmB+BGEOgelgel AmB + BGEO showing a hysteresis gelshowing showing loop. a ahysteresis hysteresisloop. loop. The Theextensibility extensibility profile ofofof profile AmB+BGEO AmB + BGEO gelgel atgel 25 25 °C ◦ C described described at °C 25 in Figurein 3Figure showed that that 3 showed The extensibility profile AmB+BGEO at described in Figure 3 showed the the extensibility thatextensibility values the extensibility values increased values proportionally increased increased to loading proportionally proportionally weight to loading to loading until reaching weightweight a maxi- until reaching until reaching a maxi-a mum extensibility maximum mum extensibility of of27.81 extensibility of cm 27.81 2 27.81 cm2 following cmfollowing 2 followinga one-phase association a one-phase a one-phase mathematical association association model. mathematical mathematical model.model. Figure 3. Extensibility profile of AmB+BGEO gel at 25 °C. AmB + BGEOgel ◦ C. gelatat2525°C. Figure 3. Extensibility profile of AmB+BGEO 2.4. Stability Studies 2.4. Stability Studies 2.4. Stability Studies The short-term stability evaluation of AmB+BGEO gel (Table 3) confirmed that after The short-term 120 days storage atstability evaluation 40 °C, the of AmB + BGEO gel (Table 3) confirmed that after The of short-term 30 °C and stability ◦ C andevaluation formulation of AmB+BGEO ◦ C, the maintained gel (Table a homogeneous 3) confirmedaspect that after 120 and days of suitable storage pH at values 30 for its 40 application on formulation skin (5.48 ± maintained 0.12 to 5.85 ± a homogeneous 0.09 and 5.12 ± aspect 0.07 120 days of storage at 30 °C and 40 °C, the formulation maintained a homogeneous aspect and suitable to 5.83 pH ± 0.07,pH values for respectively). its application on skin (5.48 ± 0.12 to 5.85 ± 0.09 and 5.12 ± 0.07 and suitable values for In itsthe same way, application onthe content skin (5.48 of AmBtopresent ± 0.12 in the 5.85 ± 0.09 formula- and 5.12 ± 0.07 to 5.83 tion ± 0.07, respectively). remained In thechanges without significant same way, the content during of of AmB the present in the formulation to 5.83 ± 0.07, respectively). In the same way, the the time content of AmB stability presentstudy in thewith a formula- remained decrease ofwithout 0.86% atsignificant 30 °C andchanges during 1.1% at 40 °C. the time of the stability study with a decrease tion remained◦without significant changes during the time of the stability study with a of 0.86% at 30 C and 1.1% at 40 ◦ C. decrease of 0.86% at 30 °C and 1.1% at 40 °C. Pharmaceuticals 2021, 14, 1033 5 of 15 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 5 of 15 Table 3. Stability studies of AmB + BGEO gel. Table 3. Stability studies of AmB+BGEO gel. 30 ± 2 ◦ C/65 ± 5% RH 40 ± 2 ◦ C/75 ± 5% RH Time (Days) 30 ± 2 °C/65 ± 5% RH 40 ± 2 °C/75 ± 5% RH Time (Days) Appearance pH Drug Content (%) Appearance pH Drug Content (%) Appearance pH Drug Content (%) Appearance pH Drug Content (%) 1 Homogeneous 5.85 ± 0.09 99.65 ± 0.08 Homogeneous 5.83 ± 0.07 99.67 ± 0.12 1 Homogeneous 5.85 ± 0.09 99.65 ± 0.08 Homogeneous 5.83 ± 0.07 99.67 ± 0.12 60 Homogeneous 5.57 ± 0.04 99.18 ± 0.43 Homogeneous 5.43 ± 0.15 98.91 ± 0.17 60 120 days Homogeneous 5.48 Homogeneous 5.57±± 0.12 0.04 99.18 ± 0.43 98.79 ± 0.66 Homogeneous 5.12 Homogeneous 5.43±±0.07 0.15 98.91 ± 0.17 98.56 ± 0.32 120 days Homogeneous 5.48 ± 0.12 98.79 ± 0.66 Homogeneous 5.12 ± 0.07 98.56 ± 0.32 2.5. In Vitro Release Study 2.5. In Vitro Release Study Figure 4 shows the release profile of AmB from the formulation. At the end of the Figure 4 shows the release profile of AmB from the formulation. At the end of the experiment, an amount of 154.8 µg of AmB was released from the gel, which represents experiment, an amount of 154.8 µg of AmB was released from the gel, which represents about 80% of the drug. The best fit of the experimental data was obtained with the about 80% of the drug. The best fit of2 the experimental data was obtained with the Boltz- Boltzmann sigmoidal model with a r = 0.9974 and whose mathematical equation is: mann sigmoidal model with a r2 = 0.9974 and whose mathematical equation is: V50−−x x   V50 Y= Y= Bottom Bottom ++(Top − Bottom (Top − Bottom) )/1 +1 exp + exp Slope (1) (1) Slope where Y is the amount of released drug, Top Top and and Bottom Bottom are are the the initial initial and and final final values values of of drug release, release, V50 V50isisthe thetime timeitittakes takestotorelease releasehalf half of the maximum amount susceptible of the maximum amount susceptible to to release release andand thethe Slope Slope of the of the curve curve indicates indicates thethe steepness. steepness. Figure 4. In In vitro vitro release release profile profile of ofAmB AmBfrom fromformulation. formulation.The Thecumulative cumulativeamount amountreleased was released was plotted against time. Data represents mean mean ±± SD (n = 3). SD (n = 3). 2.6. Ex Vivo 2.6. Ex Vivo Permeation Permeation Study Study Ex Ex vivo vivo permeation permeation studies studies of ofAmB + BGEOgel AmB+BGEO gel and and AmB AmB gel (B. graveolens gel (B. graveolens essential essential oil-free AmB gel) were carried out in order to compare and evaluate oil-free AmB gel) were carried out in order to compare and evaluate the role of the the role of the B.B. graveolens essential oil in drug permeation. AmB was not detected in the aliquots graveolens essential oil in drug permeation. AmB was not detected in the aliquots extracted extracted from the receptor from the receptorcompartment compartmentover overa a2424 h period h period of of permeation permeation assay assay in either in either of two of the the two formulations. formulations. However, However, AmB was found inside the skin with a retention AmB was found inside the skin with a retention value of 997.78 value of 997.78 µg/g skin/cm 2 for AmB + BGEO gel and 603.91 µg/g skin/cm2 for AmB gel. µg/g skin/cm2 for AmB+BGEO gel and 603.91 µg/g skin/cm2 for AmB gel. 2.7. Efficacy Study: Antimicrobial Activity 2.7. Efficacy Study: Antimicrobial Activity The antimicrobial activity after 48 h is reported in Table 4 as Minimum Inhibitory The antimicrobial activity after 48 h is reported in Table 4 as Minimum Inhibitory Concentration (MIC) values against strains of C. albicans, C. glabrata and C. parapsilosis. The Concentration (MIC) values against strains of C. albicans, C. glabrata and C. parapsilosis. B. graveolens essential oil at concentrations of 6.25 to 12.50% (v/v) showed antimicrobial The B. graveolens activity against theessential studiedoilCandida at concentrations strains. The ofAmB 6.25 to + 12.50% BGEO gel(v/v) showed the exhibited antimicro- lowest MIC values (0.29, 0.39 and 0.58 µg/mL) compared with AmB gel (0.58, 0.58 and the bial activity against the studied Candida strains. The AmB+BGEO gel exhibited lowest 1 µg/mL) MIC values (0.29, 0.39 and 0.58 µg/mL) compared with AmB against C. albicans, C. glabrata and C. parapsilosis, respectively. gel (0.58, 0.58 and 1 µg/mL) against C. albicans, C. glabrata and C. parapsilosis, respectively. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 15 Pharmaceuticals 2021, 14, 1033 6 of 15 Table 4. MIC against different cultures of Candida spp., Free AmB, AmB gel, AmB+BGEO gel and Table 4. MIC against different cultures of B. graveolens Candida essential oilspp., Freeafter (BGEO) AmB, AmB gel,atAmB incubation 30 °C+for BGEO 48 h gel 3). B. graveolens essential oil (n =and (BGEO) after2021, Pharmaceuticals incubation ◦ C for 48 h (n = 3). at 30REVIEW 14, x FOR PEER 6 of 15 MIC (µg/mL) % (v/v) Tested Species Origin FreeMIC AmB AmB Gel AmB+BGEO Gel (µg/mL) BGEO% (v/v) Tested Species Origin Table 4. MIC against different cultures of Candida spp., Free 0.58 AmB, AmB gel, AmB+BGEO gel and C. albicans ATCC Free AmB 10231 0.15 AmB Gel 0.29 AmB + BGEO Gel 12.50 BGEO B. graveolens essential oil (BGEO) after incubation at 30 °C for 48 h (n = 3). C. grabrata ATCC 66032 0.60 0.58 0.39 6.25 C. albicans ATCC 10231 0.15 0.58(µg/mL) 0.29 % (v/v) 12.50 C. grabrata ATCC C. parapsilosis Tested 66032 Species ATCC0.60 22019 Origin 0.30MIC 0.58 1 0.58 0.39 12.506.25 Free AmB AmB Gel AmB+BGEO Gel BGEO C. parapsilosis ATCC 22019 0.30 1 0.58 12.50 C. albicans ATCC 10231 0.15 0.58 0.29 12.50 2.8. Tolerance Studies C. grabrata ATCC 66032 0.60 0.58 0.39 6.25 2.8.1. Cytotoxicity C. parapsilosis Studies ATCC 22019by MTT 0.30 Assay 1 0.58 12.50 2.8. Tolerance Studies 2.8.1.The effect of different Cytotoxicity concentrations of AmB+BGEO gel on human keratinocytes was 2.8. Tolerance Studies Studies by MTT Assay evaluated using MTT cytotoxicity assay. After 24 h of incubation, it was observed that the The effect Studies 2.8.1. Cytotoxicity assayed dilutions of of different the by MTTconcentrations Assay formulation of AmB (1/50 to 1/2000) did +notBGEO affectgel cellon human viability, keratinocytes which was was evaluated The effect of using differentMTT cytotoxicity concentrations of assay. AmB+BGEO After gel on24 h human of incubation, keratinocytes close to 100% in relation to the control (Figure 5). Therefore, these results suggest it was was observed that that evaluated the assayedusing MTT cytotoxicity dilutions of the assay. After 24 h(1/50 formulation of incubation, to it wasdid 1/2000) observed not that the affect cell viability, which AmB+BGEO gel do not trigger toxicity in the cells. assayed dilutions of the formulation (1/50 to 1/2000) did not affect cell viability, which was was close to 100% in relation to the control (Figure 5). Therefore, these results suggest that close to 100% in relation to the control (Figure 5). Therefore, these results suggest that AmB AmB+BGEO+ BGEO gel gel do notdo not trigger trigger toxicity intoxicity the cells.in the cells. Figure 5. Percentage of cell viability of HaCaT cell line exposed to AmB+BGEO for 24 h at different Figure Figure 5.5. Percentage Percentage of of cellcell viability viability of HaCaT of HaCaT cellcell lineline exposed exposed to AmB + BGEO to AmB+BGEO for 24 for h at24different h at different concentrations. concentrations. concentrations. 2.8.2. In Vivo Tolerance Studies 2.8.2.No 2.8.2. InIn Vivo Vivo Tolerance Tolerance significant Studies changesStudies in the TEWL or SCH values with respect to the basal state Nosignificant significant were observed No changes after 25 min ininthe of topical changes the TEWLor application TEWL oforAmB+BGEO SCHvalues SCH values with gel in with respectarea the ventral respect to the to the basal of basal state state were the forearm after observed of the 25 volunteers min of (Figure topical 6).application These results of suggest AmB that + the formulation BGEO gel in doesventral area of the the were observed after 25 min of topical application of AmB+BGEO gel in the ventral area of not cause damage or irritation in the skin barrier. forearm the forearm of of thethevolunteers volunteers(Figure (Figure6). 6).These These results results suggest that the suggest that theformulation formulationdoes does not cause damage or irritation in the not cause15damage or irritation in the skin skin barrier. 80 barrier. 15 80 12 60 g/cm /h g/cm2 /h A.U. 12 9 40 60 A.U. 2 * * * 6 20 9 0 5 15 25 0 40 5 15 25 minutes * minutes (A) * (B) * 6 20 0 5 15 25 0 5 15 25 minutes minutes (A) (B) Figure 6. Tolerance studies in human individuals. (A) TEWL: Transepidermal water loss; (B) SCH: Stratum corneum hydration. Results are expressed as median, minimum and maximum (n = 12). Statistically significant differences: * p < 0.05. Pharmaceuticals 2021, 14, 1033 7 of 15 3. Discussion AmB is administered by intravenous infusion in the treatment of fungal infections [28]. This treatment causes several adverse effects including nausea, vomiting, rigors, fever, hypertension or hypotension, hypoxia and nephrotoxicity [16]. The incorporation of AmB into topical formulations could be used as a strategy to achieve a local effect in the skin without adverse systemic effects in the treatment of dermal candidiasis. However, its low aqueous solubility, great molecular weight (926 Da) and limited permeability of the SC to external substances, including drugs, could obstruct its penetration through the skin. In this study, the incorporation of B. graveolens essential oil into an AmB topical gel was carried out in order to promote drug permeation through the SC and its retention in human skin. The final formulation of AmB + BGEO gel (0.1%) was prepared using pharmaceutical excipients compatible with the drug (Table 2). This formulation was homogeneous with a pleasant citrus scent and had a slightly acidic pH value which assures non-irritating effects [29]. Determination of the flow properties of formulations, particularly those intended for skin application, provides important information about stability, sensory characteristics, spreadability and filling/dosing behavior [30]. A topical formulation should exhibit optimal viscosity and spreadability since a product too fluid or too viscous could be unpleasant or uncomfortable for patients [31,32]. Moreover, the viscosity and rheological behavior can modulate biopharmaceutical parameters such as release rates of drugs from their vehicles [33]. AmB + BGEO gel showed a mean viscosity of 5036 ± 45.71 mPa·s at 50 s−1 and a maximum extensibility of 27.81 cm2 , which suggest that this formulation is easy to apply (Figures 2 and 3). Rheological analysis and mathematical modeling confirmed the pseudoplastic behavior of AmB + BGEO gel and the presence of a hysteresis loop in the rheograms, suggesting thixotropic behavior [34]. These characteristics offer advantages for topical formulations since the viscosity decreases with friction, allowing for easy spreadability while returning to the initial state once the friction stops, which favors the residence in the treated area [35]. The stability studies at 30 and 40 ◦ C for 120 days showed that AmB + BGEO gel maintained its homogeneous appearance and no significant changes in its pH values were detected after storage, showing values within the suitable range for skin application. Chem- ical stability was also observed with slight yet inconsequential changes in the quantified drug content, thus demonstrating high compatibility between the drug and the excipients (Table 3). Interactions between the vehicle and drug define the release profile, which in turn affects the permeation rate of the drug [29]. In this research, the in vitro release study showed a released amount of 154.8 µg of AmB from the gel (Figure 4), which indicates that the vehicle is capable of releasing the drug without limiting skin permeation. According to the obtained r2 value, the release kinetic of AmB from gel followed a Boltzmann sigmoidal model that is characterized by an initial phase of slow-or-negligible release (0–6 h) followed by a second phase of immediate-or-controlled release [36]. Ex vivo permeation studies are used to estimate the in vivo behavior of the formula- tion, since the composition and the physical properties of the carrier influence the amount of drug reaching the target area [30]. The success of topical treatment is contingent upon the ability of the drug to cross the SC as well as its distribution through the epidermis and dermis [37,38]. The results of this study showed that the drug does not reach the receptor compartment in either of the two formulations (AmB + BGEO gel and AmB gel) but is instead retained in the tissue, thereby promoting a local effect in the skin without adverse effects. However, a higher amount of drug retained inside the skin was observed in the AmB + BGEO gel (997.78 µg/g skin/cm2 ) compared to AmB gel (603.91 µg/g skin/cm2 ), suggesting that the incorporation of B. graveolens essential oil facilitated the penetration of the drug through the SC and its diffusion to the epidermis and dermis. This essential oil contains limonene, a lipophilic terpene reported to have a skin permeation-enhancing effect of both hydrophilic and lipophilic drugs [39]. The proposed mechanism of this effect is due Pharmaceuticals 2021, 14, 1033 8 of 15 to changes in the intercellular packing and disruption in lipid structures that involve the modification of SC from solvents, thus improving drug partitioning into the skin [40,41]. The efficacy study showed that AmB + BGEO gel exhibited a lower MIC value against strains of C. albicans, C. glabrata and C. parapsilosis and is consequently more effective than the formulation sans essential oil (AmB gel). Significant antimicrobial activity of B. graveolens essential oil against strains of C. albicans has been previously reported. This effect has been attributed to its terpene-rich composition, namely limonene, although the exact mechanism is not completely understood as of yet. Nevertheless, some studies support the idea that these compounds could cause membrane disruption which consequently induces the death of the fungus [42]. A number of studies have shown that natural secondary metabolites with low molecular weight (≤500 g/mol) may act as adjuvants for antimicrobial drugs and thus potentiate its efficacy. Based on this approach, studies of combination therapy of antimicrobial drugs with terpenes have revealed promising effects against both susceptible and resistant pathogens [43]. In particular, essential oils such as Citrus aurantium, Thymus kotschanus, myrtus communis and Mentha piperita have shown a synergistic effect with clinical drugs including AmB, fluconazole, ketoconazole and meropenem [43–46]. In the current study, the combination of AmB with B. graveolens essential oil in a topical gel showed enhanced antifungal activity against C. albicans, C. glabrata and C. parapsilosis. These results indicate that the addition of B. graveolens essential oil in the formulation likely enhances the antimicrobial effect either due to its active compounds or a possible synergistic effect between its compounds and the drug. The tolerability of AmB + BGEO gel was analyzed by in vitro and in vivo models. In vitro models are considered helpful to screen the toxicity of new formulations prior to the pre-clinical and clinical assessment. A variety of cell lines are frequently used for toxicity screening in a living system due to the cells being generally easy to cultivate, fast to grow and are sensitive to toxic irritation [47]. In this study, the tested dilutions of AmB + BGEO gel did not induce relevant cytotoxic effects on cells after 24 h of incubation, which confirms high biocompatibility between the developed formulation and human keratinocytes. This result was confirmed by evaluation of biomechanical skin properties including TEWL and SCH, which allow analysis of the integrity of the skin barrier after exposure to physical or chemical agents [30,48]. The results revealed an increase in TEWL and SCH values 5 min after topical application of AmB + BGEO gel; however, a tendency to return to the basal state was observed in both parameters after 25 min of assay, suggesting that AmB + BGEO gel does not cause irritation or damage to the skin surface of volunteers. 4. Materials and Methods 4.1. Materials Amphotericin B (potency of 864 µg/mg) was purchased from Acofarma® (Barcelona, Spain). B. graveolens essential oil was provided by the Unit Operations Laboratory of the Universidad Técnica Particular de Loja [25]. Dimethyl Sulfoxide (DMSO) was obtained from Alfa Aesar (Thermo Fisher, Karlsruhe, DE-BW, Germany). Glycerin, propylene glycol 400 and castor oil were supplied by Sigma-Aldrich (Madrid, Spain). Capric Triglyceride (Labrafac™ lipophile WL 1349), polyglyceryl-3 dioleate (Plurol® oleique CC497), diethy- lene glycol monoethyl ether (Transcutol® P), and propylene glycol monolaurate-type II (Lauroglycol™ 90) were obtained from Gatefossé (Saint-Priest, France). Carboxymethylcel- lulose (CMC), carbopol® 940, sodium benzoate, parabens, and citric acid were obtained from Fagron Ibérica (Barcelona, Spain). HaCaT cell line was purchased from Cell Line Ser- vices (Eppelheim, DE-BW, Germany) and the reagents used for cell cultures were obtained from Gibco (Carcavelos, Lisbon, Portugal). The reagents for the MTT assay were obtained from Invitrogen Alfagene® (Carcavelos, Lisbon, Portugal). A Millipore Milli-Q® water purification system (Millipore Corporation; Burlington, MA, USA) was used. Finally, the chemicals and reagents were of analytical grade. Pharmaceuticals 2021, 14, 1033 9 of 15 4.2. High-Performance Liquid Chromatography (HPLC) AmB was quantified by a previously validated HPLC method [49]. The assay was carried out using a Waters HPLC with 2487 (UV/Vis) Detector & 717 Plus Autosampler (Waters, Milford, MA, USA). The mobile phase was a mixture of acetonitrile and glacial acetic acid 3.75% (65:35, v/v) filtered with a 0.45 µm PVDF membrane filter (Millipore Corp., Madrid, Spain). The assay was performed using a Kromasil C18 column (250 mm, 4.6 mm and 5 µm). The mobile phase was pumped at a flow rate of 0.5 mL/min and the injection volume was 10 µL. Finally, the elute was analyzed at 407 nm (wavelength of maximum absorbance of AmB). 4.3. Solubility Studies The solubility of AmB in various solvents including DMSO, glycerin, propylene glycol 400, castor oil, Labrafac™ lipophile WL 1349, Plurol® oleique CC497 and Lauroglycol™ 90 was evaluated using an excess of AmB and mixing by magnetic stirring for 30 min at 1500 rpm. The samples were equilibrated for 24 h and subsequently centrifuged at 9000 rpm for 10 min. The supernatant was extracted and diluted with methanol in order to quantify the dissolved AmB using a UV-Visible DR 6000 spectrophotometer (Hach® , Düsseldorf, DE-NW, Germany). 4.4. Formulation: Design and Analysis of 23 Factorial Experiment In this study, a 23 factorial experiment was designed to examine the influence of three factors: the type of polymer (A), humectant (B) and preservative (C) at two levels, which are indicated by the signs “+” and “−” in Table 5 [50]. Table 5. Factors studied in the factorial experimental design and their levels. Levels Factors (+) (−) A: Polymer Carboxymethylcellulose Carbopol B: Cosolvent Glycerin Propylene glycol 400 C: Preservative Sodium benzoate Parabens Eight different formulations were obtained with the 23 factorial experiment using the ingredients at concentrations in accordance with the Handbook of Pharmaceutical Excipi- ents and published data about efficacy and safety [51,52]. Furthermore, each formulation was composed of AmB (0.1%), BGEO (2%), DMSO (5%) and purified water. The pH was adjusted using citric acid (0.25%) in the CMC formulations and triethanolamine (1%) in the carbopol 940 formulations (Table 6). Table 6. Formulations developed from the 23 factorial experiment. Component % F1 F2 F3 F4 F5 F6 F7 F8 CMC 3 * * * * Carbopol 940 1 * * * * Glycerin 20 * * * * Propylene glycol 400 20 * * * * Sodium benzoate 0.02 * * * * Parabens 0.02 * * * * Citric acid 0.20 * * * * Triethanolamine 1 * * * * AmB 0.1 * * * * * * * * DMSO 5 * * * * * * * * BGEO 2 * * * * * * * * Water sq * * * * * * * * BGEO = Bursera graveolens essential oil, CMC = Carboxymethylcelullose, AmB = Amphotericin B, DMSO = Dimethyl Sulfoxide, sq = Sufficient quantity, * presence of the component (featured in the left-most column). Pharmaceuticals 2021, 14, 1033 10 of 15 In order to prepare the gels, polymers were hydrated for 30 min in water (mix 1). AmB was dissolved in DMSO under magnetic stirring for 10 min immediately followed by adding cosolvent and B. graveolens essential oil (mix 2). Citric acid and preservatives were dissolved in water (mix 3). Finally, mix 1 and 3 were mixed at 850 rpm using a IKA ULTRA-TURRAX T50 (Staufen, Germany) for 10 min, after which mix 2 was incorporated under the same stirring conditions. The final formulation was selected based on physical and chemical stability after 60 days of storage at 30 ◦ C. 4.5. Characterization of AmB + BGEO Gel The pH of AmB gel was determined using a pH meter GLP 22 (Crison Instruments, Barcelona, Spain). Viscosity and rheological behavior of AmB gel were evaluated with a Haake Rheostress 1 rotational rheometer (Thermo Fisher Scientific, Kalsruhe, Germany) equipped with a cone–plate sensor system including a fixed bottom plate and a Haake C60/2Ti movable top cone (60 mm diameter, 2◦ angle, 0.105 mm gap). AmB gel was tested in duplicate 24 h after preparation. Viscosity values and flow curves were recorded during the ramp-up period from 0 to 50 s−1 (3 min), constant shear rate period of 50 s−1 (1 min), and a ramp- down period from 50 to 0 s−1 (3 min). Obtained data from the flow curve were fitted to different mathematical models including Newtonian, Bingham, Ostwald-de-Waele, Cross, Casson and Herschel–Bulkley. The model that best statistically describes the experimental data was selected according to the correlation coefficient value (r). Viscosity mean value (mPa·s) was determined at 50 s−1 from the constant shear rate period of viscosity curve. The determination of the disturbance of the microstructure during the test or “apparent thixotropy” (Pa/s) was evaluated by determining the area of the hysteresis loop. The extensibility of AmB gel was analyzed in triplicate following the previously described method [53]. A sample of 1 g of formulation was placed on the center of the base plate of an extensometer. Afterwards, a glass plate (7.93 g) was placed on the sample without sliding and a series of weights (27.89, 57.82, 107.69, 157.55, 207.46, 237.50 and 307.69 g) were added at 1 min intervals. The extended area of the sample was recorded and then fitted to mathematical models using GraphPad Prism® version 6.0 (GraphPad Software Inc., San Diego, CA, USA). The drug content in the gel was determined by dissolving 100 mg of AmB gel in 5 mL of DMSO:methanol (1:1, v/v). After vortexing for 2 min, the solution was filtered and analyzed by HPLC method described in Section 4.2. 4.6. Stability Studies After manufacturing, AmB + BGEO gel samples were stored at pre-established con- ditions of temperature and relative humidity (RH) in accordance with the Q1A(R2) ICH (International Conference on Harmonisation) Guidelines: 30 ± 2 ◦ C/65 ± 5% RH and 40 ± 2 ◦ C/75 ± 5% RH for 4 months [54]. Physical and chemical characterization of the formulation were carried out before and after storage in the aforementioned conditions using the method described in Section 4.5. 4.7. In Vitro Release Study The release study of AmB from gel was performed using Franz diffusion cells of 13 mL and an effective diffusion area of 2.54 cm2 (FDC 400; Crown Grass, Somerville, NJ, USA). The receptor medium (RM) was a mixture of NN-dimethyl formamide, methanol and water (55:5:40, v/v). The conditions of the experiment were maintained at 32 ◦ C and under continuous stirring in order to achieve sink conditions. A 0.45 µm nylon membrane was mounted between the donor and receptor compartments. A sample of 200 mg of AmB + BGEO gel was placed in the donor compartment and aliquots of 300 µL were collected from receptor compartment and replaced with the same volume of fresh RM at predetermined time intervals (2, 4, 6, 18, 21 and 24 h). The released amount of AmB (µg) from the formulation was determined by HPLC (Section 4.2) and plotted versus time (h) Pharmaceuticals 2021, 14, 1033 11 of 15 using GraphPad Prism® 6.0 (GraphPad Software Inc., San Diego, CA, USA, 2014). The experiment was carried out in triplicate and data are represented as mean ± SD. Data from the release curve were fitted to several kinetic models including first order, Higuchi, Hyperbolic, Weibull and Korsmeyer–Peppa. Finally, the model with the highest coefficient of determination (r2 ) was selected. 4.8. Ex Vivo Permeation Study The permeation studies were carried out using AmB + BGEO gel and AmB gel (B. graveolens essential oil-free AmB gel developed with the same qualitative and quan- titative formula only without the incorporation of the essential oil) in order to compare and evaluate the role of the B. graveolens essential oil in drug permeation. These ex vivo permeation studies were carried out in Franz diffusion cells of 6 mL (0.64 cm2 diffusion area) using human skin obtained during an abdominal lipectomy of a healthy 38-year-old woman (Hospital of Barcelona, SCIAS, Barcelona, Spain). To that end, a written informed consent was provided by the volunteer in accordance with the Ethical Committee of the Hospital of Barcelona (number 001, dated 20 January 2016). To guarantee the integrity of the skin samples, Transepidermal water loss (TEWL) was measured using a Tewameter TM 300 (Courage & Khazaka Electronics GmbH; Cologne, Germany) and only those with results below 10 g/m2 h were used. Skin samples (0.4 mm thick) were mounted between the donor and receptor compartments. Transcutol® P was used as receptor medium (RM) which was kept at 32 ◦ C and under stirring to guarantee sink conditions. A sample of 200 mg of AmB + BGEO gel or AmB gel was placed in the donor compartment in contact with the outer surface of skin. Aliquots of 300 µL were collected from the receptor compartment and replaced with the same volume of fresh RM at predetermined time intervals (2, 6, 18, 24, 28, 45 and 50 h). These aliquots were analyzed by HPLC (Section 4.2). Following per- meation studies, these skin samples were removed from the Franz diffusion cells, washed with distilled water and cut along the edges in order to retain only the permeation area. Afterwards, skin samples were weighed and immersed in 1 mL of DMSO during 20 min under cold sonication using an ultrasonic bath in order to extract AmB retained in the skin (Qret, µg drug/g tissue/cm2 ). Finally, the resulting solution was filtered and analyzed by HPLC. 4.9. Efficacy Study: Antimicrobial Activity The efficacy of the formulation was evaluated by determination of Minimum In- hibitory Concentration (MIC), which is defined as the lowest concentration of an antimicro- bial agent necessary to inhibit the growth of a microorganism. The MIC was calculated by the broth microdilution method against three different strains of Candida spp. including C. albicans ATCC 10231, C. glabrata ATCC 66032 and C. parapsilosis ATCC 22019 (American Type Culture Collection, Manassas, VA, USA). This assay was carried out according to the guidelines outlined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the CLSI Reference Method M27-A3 [55,56]. A synthetic medium containing RPMI-1640, glutamine, pH indicator without bicar- bonate and glucose 2% w/v: RPMI-1640 2% G (Invitrogen, Madrid, Spain) was used as culture medium. The yeast strains were first cultured on a Sabouraud Dextrose Agar Medium (Invitrogen, Madrid, Spain) at 30 ◦ C for 48 h. The inoculums were prepared by suspending yeast colonies in sterile 14 Ringer’s solution to achieve a density equivalent to 0.5 McFarland standards and counting in a Neubauer Chamber (1 to 5 × 106 Colony Form- ing Unit, CFU/mL). Subsequently, a 1:10 dilution (1 to 5 × 105 CFU/mL) was prepared to be used as the final inoculum. A solution of AmB previously dissolved in DMSO (Free AmB) as well as samples of AmB + BGEO and AmB gel (B. graveolens essential oil-free AmB gel) were evaluated to perform a comparative analysis. The inoculum was used as a positive control and the culture medium as a negative control. Pharmaceuticals 2021, 14, 1033 12 of 15 The experiment was performed using 96-well microdilution plates making serial double dilutions of the samples under analysis from 37.5 µg/mL to 0.0002 µg/mL for the formulations and 100% to 0.04% for the B. graveolens essential oil. Finally, 100 µL of the inoculum was added. Plates were read at t 0, 24 and 48 h after incubation at 30 ◦ C with a microplate reader model 680 (Bio-Rad, Madrid, Spain) at 620 nm. 4.10. Tolerance Studies 4.10.1. Cytotoxicity Studies by MTT Method In vitro MTT cytotoxicy assay were performed using the human keratinocytes cell line HaCaT. Cells were adjusted at 2 × 105 cell/mL, seeded in 96-well plate for 24 h in Dubelcco’s Modified Eagle’s Medium (DMEM) with high glucose content buffered with 25 mM HEPES, and supplemented with 1% non-essential amino acids, 100 U/mL penicillin, 100 g/mL streptomycin and 10% heat inactivated Fetal Bovine Serum (FBS). Next, the cells were incubated with different dilutions of the AmB + BGEO gel ranging from 1/50 to 1/2000 for 24 h. Later, the HaCaT cells were washed with 1% sterile PBS and incubated with MTT (Sigma-Aldrich Chemical Co, St. Louis, MO, USA) solution (2.5 mg/mL) for 2 h at 37 ◦ C. The medium was then carefully removed and 0.1 mL of solubilization reagent (99% DMSO) was added to lyse the cells and dissolve the purple MTT crystals. Cell viability was measured at 570 nm in a microplate photometer Varioskan TM LUX (Thermo Scientific, Waltham, MA, USA). A negative control was processed in parallel for comparison. The results were expressed as percentage of cell survival relative to the control (untreated HaCaT cells; 100% viability) using the following equation: Abs sample % Cell viability = × 100 (2) Abs control 4.10.2. In Vivo Tolerance Measurements of transepidermal water loss (TEWL) and stratum corneum hydration (SCH) of the ventral area of the forearm of 12 volunteers (6 men and 6 women; ages between 20 and 35 years) with healthy skin were performed with prior written informed consent. This study was approved by the Ethics Committee of the University of Barcelona (IRB00003099) in accordance with the principles from the Declaration of Helsinki. The time intervals of each measurement were 5, 15 and 25 min after absorption of AmB + BGEO gel in the application area. Readings were recorded using a Tewameter® TM300 and Corneometer® 825 (Courage & Khazaka Electronics GmbH, Cologne, Germany) for TEWL and SCH, respectively. For TEWL measurements, the probe was pressed and held on the skin for 2 min and the results are expressed as g/cm2 /h. For the SCH values, the probe was pressed on the skin to measure the dielectric constant of the skin where measurements were given in arbitrary units (AU). The results of TEWL and SCH were recorded as the median, minimum and maximum (n = 12). 5. Conclusions In conclusion, the present study provides evidence that the topical gel of AmB en- riched with B. graveolens essential oil offers appropriate characteristics for skin application including biocompatible values of pH, pseudoplastic behavior, optimal spreadability and acceptable tolerability, all of which make it an appealing and suitable formulation for human use. Furthermore, the incorporation of B. graveolens essential oil improved the biopharmaceutical profile of the drug, facilitating its penetration through the SC and its retention into the skin and consequently promoting a local effect in the target area linked to enhanced antifungal activity against C.albicans, C. glabrata and C. parapsilosis. Therefore, this formulation could constitute a promising alternative in the treatment of cutaneous candidiasis which encourages further research to explore its use in clinical practice. Author Contributions: L.C.E. carried out all the experiments, analyzed the data/results, and wrote the paper; P.C.G. realized the in vivo tolerance studies and wrote the paper; L.S. carried out the Pharmaceuticals 2021, 14, 1033 13 of 15 biopharmaceutical and rheological studies and wrote the paper; N.B. developed the antimicrobial activity studies; N.D.-G. realized the cytotoxicity assay; B.C.-T. carried out the 23 factorial experiment; and A.C.C. conceived and designed all the experiments. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board (or Ethics Committee) of University of Barcelona (IRB00003099, dated 30 January 2019). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Data is contained within the article. Acknowledgments: L.C.E. acknowledges the support of the Universidad Técnica Particular de Loja and the Secretaría de Educación Superior, Ciencia, Tecnología e Innovación (SENESCYT—Ecuador). The authors would like to express their gratitude to Eduardo Valarezo for his provision of the B. graveolens essential oil. Finally, the authors also acknowledge Jonathan Proctor for his review of the use of the English language. Conflicts of Interest: The authors declare no conflict of interest. References 1. Watts, C.J.; Wagner, D.K.; Sohnle, P.G. Fungal Infections, Cutaneous. In Encyclopedia of Microbiology, 3rd ed.; Elsevier: New York, NY, USA, 2009; pp. 382–388. 2. Havlickova, B.; Czaika, V.A.; Friedrich, M. Epidemiological Trends in Skin Mycoses Worldwide. Mycoses 2008, 51, 2–15. [CrossRef] 3. Permana, A.D.; Paredes, A.J.; Volpe-Zanutto, F.; Anjani, Q.K.; Utomo, E.; Donnelly, R.F. 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