Topical Delivery of Amphotericin B Utilizing Transferosomes for the Treatment of Cutaneous Leishmaniasis

: Amphotericin B (AmB) is a high-molecular weight poorly soluble drug with a high efficacy in the treatment of infectious caused by Leishmania spp. parasites, which possesses a very low topical bioavailability. Transferosomes (TFs) are ultradeformable vesicles that consist of drugs lipids, an edge activator, and a low amount of ethanol (<10%). They have been engineered and optimized to enhance the permeation of AmB across the skin and, thus, its antiparasitic activity. Drug loading of the formulation resulted in 0.086%, while a good physicochemical stability for 6 months under desiccated conditions was observed. AmB-TFs illustrated a flux of 4.91 ± 0.41 µg/cm 2 /h across mouse skin. In vivo studies demonstrated a good permeation of the drug after topical application on healthy mouse skin, which was increased using microneedles at early exposure times, while in vivo efficacy studies demonstrated that the parasite load was decreased in a 98.24 ± 1.54%.


Introduction
Nowadays, cutaneous leishmaniasis remains a common public health problem due to the lack of an effective and safe treatment to ensure healing and prevent parasite dissemination, while also avoiding undesired systemic effects. The main challenge resides in the utilization of parenteral and oral treatments, which should be suitable for visceral and mucocutaneous leishmaniasis but become highly toxic in the treatment of skin infections. Amphotericin B (AmB) is a macrolide which possesses a high molecular weight (924 g/mol) and is naturally synthetized by the fungi Streptomyces nodosus [1,2]. This molecule has a broad spectrum antifungal drug, which also presents antileishmanial activity due to its ability to bind the ergosterol in cell membranes [1]. However, the systemic utilization of AmB would have an undesirable result, especially considering its narrow therapeutic window [3], but also due to its low solubility in aqueous media. As a result, AmB could present a wide variety of adverse effects, but also a poor permeability across biological barriers [1,4].
Transferosomes (TFs) are lipid vesicles that consist of a mixture of drug, lipids, and edge activators with reduced ethanol content (below 10%). They are capable of squeezing themselves and losing their original structure to permeate across the skin [5,6]. Thus, TFs can enhance the permeability of poorly soluble drugs, such as AmB, promoting drug targeting, and reducing the risk of undesired effects [5,6]. In this work, AmB-TFs were engineered and optimized via quality by design (QbD) tools to ensure a high entrapment efficiency (EE), appropriate particle size distribution, and good colloidal and physicochemical stability. This formulation was in vitro and in vivo tested to understand drug permeation across synthetic and biological membranes, but also to better understand the efficacy of this drug delivery system in Leishmania amazonensis and L. braziliensis promastigotes and amastigotes. In vitro toxicity was also evaluated in terms of cytotoxicity and hemolysis to ensure the safety of the developed formulation.

Optimization and Manufacturing of AmB-TFs
A three-level Box-Behnken model was employed to optimize the AmB-TFs formulation. DesignExpert 10 software (Stat-Ease Inc., Minneapolis, MN, USA) was used to establish the high and low levels of the design. Selected independent and dependent variables and their ranges are shown in Table 1.
AmB-TFs were manufactured using the thin film hydration method. First, 1.82 mg of AmB, 171.74 mg of Lipoid S100, and 28.26 mg of NaDC were dissolved in 20 mL of methanol in a round-bottom flask. The solvent was evaporated using a RV 06-ML rotaevaporator (Janke and Kunkel Ika-Labortechnik, Staufen, Germany) and a Vac V-500 vacuum pump (Büchi Labortechnik, Flawil, Switzerland) at 40-45 °C and 250 rpm. After evaporation, a film was obtained and reconstituted using a mixture of ethanol and phosphate buffered saline (PBS) at pH 7.4 (91.5:8:5 v:v). The mixture was homogenized for 30 min and then probe sonicated at 10 W (Branson Sonifier 250, Emerson Industrial, East Granby, CT, USA). Solid-state AmB-TFs were also obtained via freeze-drying. Mannitol was added to liquid AmB-TFs as a cryoprotectant agent. Samples were frozen at −40 °C overnight and freeze-dried at −50 °C and 0.2 bar for 24 h utilizing a LyoQuest freeze-drier (Azbil Telstar, S.L., Terrasa, Spain).

Characterization of AmB-TFs
AmB-TFs were characterized in terms of particle size, zeta-potential, and EE. A Zetatrac Ultra Analyzer (Microtrac Inc., Montgomery, PN, USA) was employed to measure particle size and zeta-potential via dynamic light scattering (DLS).
On the other hand, AmB-TFs were centrifuged (6000 rpm, 15 min) to separate entrapped and unencapsulated AmB. The supernatant was diluted using methanol (1/100 v/v) and, then, absorbance was measured via UV-visible spectrophotometry (PharmaSpec, Shimadzu, Kyoto, Japan) at 406 nm. EE was calculated as follows (Equation (1)): where A was the absorbance at 406 nm immediately after probe sonication and B was the absorbance at 406 nm after centrifugation.

Physicochemical Stability of AmB-TFs
Freshly prepared liquid AmB-TFs were diluted using PBS pH 7.4 and later divided into different vials containing 1 mL of sample each. Half of the vials were freeze-dried as above described. Then, both batches were stored at 4 and 25 °C, while relative humidity (RH) was also controlled for the solid-state AmB-TFs (10 and 60% RH) using different saturated salt solutions. For this purpose, uncapped vials were placed into stability chambers at the above-mentioned conditions, and samples were analyzed at different time points. Samples were reconstituted using 1 mL of deionized water to measure particle size (physical stability) and with 1 mL of HPLC mobile phase (chemical stability) [7].

In Vitro Permeability Studies
Diffusion studies took place using AmB-TFs and AmB dissolved in dimethyl sulfoxide (DMSO). Franz cells (Soham Scientific, Loughborough, UK) were employed to understand in vitro permeation through two different membranes: synthetic Strat-M ® membranes (Merck KGaA, Darmstadt, Germany) and mouse skin. These membranes were located on top of the receptor compartment, while PBS pH 7.4 supplemented with 4% w/v NaDC was placed in the receptor compartment to ensure sink conditions. AmB concentration at different time points was quantified using a previously validated the HPLC method [7] (Jasco Inc., Easton, MD, USA).
Once the study was finished, the remaining amount of AmB was extracted and analyzed by the above described HPLC method [7].

Ex Vivo Red Blood Cells (RBCs) Haemolysis Studies
RBCs were taken from blood samples from a human donor. The process took place as previously described with some modifications [8]. Cells (4%) were diluted using PBS pH 7.4 and placed in a 96-well plate followed by AmB-TFs at different concentrations and compared with blank TFs and AmB-DMSO, while PBS pH 7.4 and Triton ® -X 100 were used as negative and positive controls, respectively. Results of hemolytic toxicity were expressed as HC50.

In Vitro Antiparasitic Activity and In Vitro Macrophage Cytotoxicity
Leishmania amazonensis and L. braziliensis were cultured in Schneider's insect medium (Sigma-Aldrich, St. Louis, MO, USA) at 26 °C supplemented with 20% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich), penicillin (100 IU/mL), and streptomycin (100 µg/mL) in culture flasks. Two different antiparasitic activity studies were carried out for promastigotes and amastigotes, as previously described [9]. Cytotoxicity was also evaluated in J774 murine macrophages following previously described protocols [9,10]. Results of efficacy and toxicity were expressed as IC50 and CC50, respectively.

In Vivo Pharmacokinetics and Pharmacodynamics
Both experiments were carried out in CD-1 male mice (8 weeks-old, 28 g). All in vivo processes, as well as approved ethical committees, are described in more detail in an already published article from the research group [2].

Optimization and Characterization of AmB-TFs
The Box-Behnken model determined that higher amounts of AmB and NaDC provided a larger particle size, while the smallest particle size would be achieved with low to intermediate amounts of NaDC and higher amounts of AmB. The largest EE was achieved when intermediate amounts of edge activators and drugs were utilized. However, none of the tested variables showed a high impact on zeta-potential.
The optimized formulation resulted in a 14:86 (w:w) edge activator: lipids ratio, with a final drug loading of 0.086%, while the reconstitution buffer consisted of 91.5% PBS pH 7.4 and 8.5% ethanol. This formulation had a particle size of 149 ± 22 nm, showed good colloidal stability with a zeta-potential of −35.02 ± 2.71 mV, and an EE of 93.3 ± 1.9%.

Physicochemical Stability of AmB-TFs
AmB-TFs remained physicochemically stable for at least one month in the liquid state at 4 °C, while freeze-dried AmB-TFs were stable for at least 6 months at 4 and 25 °C under desiccated conditions (10% RH). However, the stability of AmB-TFs was highly affected at 60% RH.

In Vitro Permeability Studies
Both AmB-TFs and AmB-DMSO showed a linear permeation of the drug across Strat-M ® membranes, with a lag time of 9.99 ± 4.59 and 4.86 ± 2.87 min and a steady-state flux value of 41.18 ± 1.39 and 211.06 ± 15.33 µg/cm 2 /h for AmB-TFs and AmB-DMSO, respectively, which means that AmB-DMSO had a 5-fold higher permeation across synthetic membranes. However, the behavior of AmB was very different when permeation was evaluated across mouse skin. The drug did not show any lag time as it started to permeate immediately, while the flux resulted in 4.91 ± 0.41 and 5.49 ± 1.18 µg/cm 2 /h for AmB-TFs and AmB-DMSO, respectively, indicating there are no significant differences when permeation is evaluated across biological barriers (Figure 1a). This would suggest that testing in vitro permeation through mouse skin should be more reliable compared with in vivo results.

Evaluation of Hemolytic Toxicity
Hemolytic studies concluded that AmB-TFs showed 10-fold higher values of HC50 than AmB dissolved in DMSO. This would suggest that entrapping AmB with TFs reduces drug toxicity, while the utilized excipients are safe for RBCs (Figure 1b).

In Vitro Antiparasitic Activity and Cytotoxicity against Macrophages
The IC50 values of AmB-TFs against promastigotes were 2.00 ± 0.24 µM and 3.42 ± 0.15 µM for L. amazonesis and L. braziliensis, respectively, while higher IC50 values (5.37 ± 0.19 and 7.08 ± 0.34 µM) were observed for the amastigotes. The CC50 against J774 murine macrophages was 33.98 ± 4.02 µM, indicating an appropriate selectivity index of AmB-TFs for both species of Leishmania.

In Vivo Pharmacokinetics and Pharmacodynamics
AmB-TFs were able to permeate to deeper regions on the skin. The application of microneedles before the topical administration of the formulation resulted useful to increase drug permeation during the first hours, especially in the deepest layers. The utilization of these solid metallic microneedles formed transient micropores that enhanced drug permeability. However, micropores tend to reseal within 2 h when the skin is not occluded, indicating microneedles could not result useful at later times (Figure 2a).
Regarding in vivo efficacy, the administration of AmB-TFs once a day for 10 consecutive days resulted in a decrease of parasite load of 98.24 ± 1.54%, which resulted in similar to the intralesional administration of Glucantime ® (99.78 ± 0.31%) (Figure 2b). The utilization of AmB-TFs could result in many advantages taking into account that the administration of pentavalent antimonials has been described as painful, but they can also produce severe adverse effects, including cardiotoxicity, pancreatitis, and nephrotoxicity [11].

Conclusions
AmB-TFs enabled the permeation of AmB after topical administration that allowed therapeutically relevant amounts to be up taken and accumulated within the dermis where parasites accumulated. The low toxicity of the formulation allowed for safe and effective non-invasive formulations for the treatment of these parasitic infections.