Topical delivery is preferable over systemic delivery for cutaneous leishmaniasis (CL), because of its easy administration, reduced systemic adverse effects, and low cost. Nitazoxanide (NTZ) has broad-spectrum activity against various parasites and has the potential to avoid drug resistance developed by enzymatic mutations. NTZ oral formulation is being associated with severe dyspepsia and stomach pain. Herein, NTZ-transethosomes (NTZ-TES) were prepared and loaded into chitosan gel (NTZ-TEG) for topical delivery. NTZ-TES were prepared by thin-film-hydration method and optimized statistically via Box-Behnken method. The optimized formulation indicated excellent particle size (176 nm), PDI (0.093), zeta potential (-26.4 mV) and entrapment efficiency (EE, 86%). The TEM analysis showed spherical sized particles and FTIR analysis indicated no interaction among the excipients. Similarly, NTZ-TEG showed optimal pH, desirable viscosity and good spreadability. NTZ-TES and NTZ-TEG showed prolonged release behavior and higher skin penetration and deposition in epidermal/dermal layer of skin in comparison with the NTZ-dispersion. Moreover, NTZ-TES showed higher percentage inhibition, lower IC50 against promastigotes and higher macrophage uptake. Additionally, skin irritation and histopathology studies indicated the safe and non-irritant behavior of the NTZ-TEG. The obtained findings suggested the enhanced skin permeation and improved anti-leishmanial effect of NTZ when administered as NTZ-TEG.

Preparation of transethosomes

The transethosomes were prepared by thin film hydration method described by Garg et al. with few modifications. For the preparation of blank transethosomes, the specified amounts of phospholipon 90 G (PL90G) and span 80 (S80) were added in 1:1 v/v blend of organic solvents i.e. chloroform and methanol and placed in the rotary flask. Rotary evaporator was used to remove the organic solvents at 50 ºC and 100 rpm. The hydration of deposited thin film was performed by using 10 mL of ethanolic-PBS (containing 20-40% (v/v) ethanol), at 60 ºC and 100 rpm for 1 hr. The obtained vesicles were then kept aside for another half hour to hydrate completely and then subjected to size reduction process by passing through 0.22 and 0.45 nm polycarbonate membrane syringe filters.

Similarly for the preparation of NTZ-TES, the NTZ was added in methanol and sonicated for 5 min, while PL90G and S80 were added to chloroform. Both the solutions were then combined in round bottom flask and organic solvents were then removed by rotary evaporator under reduced pressure.

Preparation of nitazoxanide loaded transethosomal gel (NTZ-TEG)

In order to make the prepared transethosomes rheologically acceptable the NTZ-TES were incorporated into chitosan gel (2% w/v). For the 3 mL preparation of NTZ-TEG, 100 mg of low or medium molecular weight chitosan was dissolved in 1% (v/v) acetic acid solution with continuous stirring and then 5 mL final volume was made up by adding 2 mL of PBS 7.4 having optimized NTZ-TES pellets dispersed in it. The mixture was stirred continuously till formation of homogeneous gel.

For the optimization of blank transethosomes, a two-level three-factors, Box-Behnken Design was utilized by employing a software named as Design Expert® (Version 12, Stat-Ease Inc., USA).The independent variables selected were lipid, surfactant and ethanol while dependent variables were particle size, PDI and zeta potential.

NTZ-TES PS, PDI, ZP determination and TEM analysis

Dynamic light scattering (DLS) method was employed to characterize NTZ-TES in terms of PS, PDI and ZP using the Zetasizer (Malvern Zetasizer, ZS 90). Each measurement was recorded in triplicate (mean ± S.D.). The scanning was performed at 25 ºC and 90º scattering angle. While ZP was determined by the use of universal-dip-cell in the Zetasizer ZS 90

The superficial morphology of NTZ-TES was examined by using the transmission electron microscopy (TEM). In this analysis the prepared NTZ-TES formulation was placed on the grid of copper coated with carbon, in droplet form and allowed to stand for some time so that it got stick to the grid. After that a 1% phosphotungstic acid was used as the negative stain and TEM analysis was then performed at 100 kV. 

Entrapment efficiency (EE)

The indirect method was being employed to determine the EE of transethosomes. The prepared NTZ-TES were subjected to centrifugation at 15000 rpm for the period of 2 hr. After the centrifugation, the supernatant was separated from pellets, diluted with the acetonitrile and then UV-Visible spectrophotometric analysis was done to measure free NTZ. The following equation was then employed for the calculation of % EE.

%EE= Total Amount of drug- unentraped drug/total amount of drug x100

Compatibility study of drug and excipients

In order to determine the compatibility of drug and excipients, the FTIR analysis of the pure NTZ, physical mixture (PM), PL90G, and optimized NTZ-TES was performed. About 10 mg of sample was compressed along with 200 mg of KBr into a disc using a hydraulic press and FTIR spectra was measured over the range of 500-4000 cm-1 using FTIR spectrophotometer. 

The color, appearance and homogeneity of both the blank and NTZ-TEG was evaluated visually.  A digital pH-meter was used for the measurement of gel pH, for which 100 mg of the optimized gel was added to the 50 mL of distilled water and stirred to dissolve completely.  The viscosity of the both the blank and drug-loaded gel was determined by using Brookfield cone and plate rheometer at room temperature using spindle CPA 52 Z.

For measurement of gel spreadability, an area of 2 cm was marked on the glass slide and gel of 500 mg was placed. Then one more glass slide along with 500 g weight was placed on first glass slide having gel on it for 5 min at a room temperature to allow proper spreading of gel. After that, the increase in area of gel spread was measured and the percentage spreadability of gel was calculated by following equation.

% Spread area = Final area after spreading/2 Cm X 100

In vitro release and kinetic drug release study

For investigation of the NTZ release behaviour, the in vitro study was performed for NTZ-dispersion, NTZ-TES, and NTZ-TEG by using the PBS of pH 5.5 as the release medium. The pH 5.5 depicts the pH of the macrophage endosomes and also the skin. The NTZ-dispersion, NTZ-TES and NTZ-TEG having equivalent amount of drug were added into the dialysis bag. These dialysis bags were then placed in water-bath-shaker having release media with controlled temperature of 37 ± 0.5 ºC. The samples after predetermined time were taken out and replaced with the fresh PBS solution to maintain sink conditions. The drug concentration in the samples was determined by using UV spectrophotometer at 335 nm in triplicate after diluting the samples with predetermined ratio of acetonitrile. The cumulative amount of the drug released was then calculated by using following formula :

n= Vr X Cn + ∑. Vs X Ci

Ex vivo permeation and skin deposition study

For the ex vivo permeation study Franz diffusion cell apparatus was used. The rat skin was excised and clipped between the donor and receiver compartment with upward facing the stratum corneum (SC). In receiver compartment, the PBS (pH 7.4) was filled and stirring at 300 rpm was kept fixed. The NTZ-dispersion, NTZ-TES and NTZ-TEG were then placed in the donor compartment and 24 hr study was performed. The temperature of 32 ± 1 ºC was maintained throughout the experiment to simulate the skin conditions. The samples were then withdrawn at predetermined time and analyzed for NTZ using UV spectrophotometer. 

The steady state flux (Jss), the enhancement ratio (ER) and permeability coefficient (Kp) were determined by employing following equations.

Jss= Amount of permeated drug/Time

ER = Jss of the formulation/ Jss of the pure drug dispersion 

KP = Jss/ Initial amount of NTZ in donor cell

In vitro leishmanicidal activity against promastigotes

In vitro antileishmanial activity was carried out by using diphenyl-tetrazolium (MTT) colorimetric assay. The parasites were incubated in the M-199 medium having 1% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 µg/mL streptomycin-sulphates at 24 ºC. The promastigotes were counted in Neubauer hemocytometer and were seeded at the concentration of 1×10⁶ promastigotes/mL in each well of 96-well plate having 20 µL samples, at the concentration of 100 µg/mL (having <1% DMSO in PBS). 1% DMSO and amphotericin B (µg/mL) in PBS were employed as negative and positive control respectively. Incubation of these culture plates was done for the period of 72 hr at the temperature of 24 ºC. After this the sterilized, pre-filtered MTT solution at the concentration of 4 mg/mL in distilled water was prepared, added to the culture plates and incubated for 4 hr at 24 ºC. After that, the supernatant was carefully removed without disturbing the sediment having formazan crystals. For the dissolution of formazan crystals DMSO (100 µL) was added in the sediment. After 1 hr the absorbance was measured at the wavelength of 540 nm using the microplate reader. The IC₅₀ was calculated by using GraphPad prism software.  The percentage growth inhibition was then calculated by following equation

^{% Growth Inhibition = Optical density of control - optical density of test / optical density of control X 100}

Ex vivo cell uptake study

The resident macrophages were collected from the peritoneal cavity of the rats by injecting the cRPMI (cold Roswell Park Memorial Institute) media. Moreover,  the media containing these macrophages was withdrawn slowly and placed on ice. The collected suspension was centrifuged at 4 ºC for 10 min. The obtained pellets were then dispersed in the cRPMI media containing 10% FBS (Fetal bovine serum) and 3% penicillin/streptomycin. The cells were counted using hemocytometer and placed in the 96-well plate at the concentration of 2 × 10⁴ million cells/well and incubated for 24 hours with 5 % CO₂ supply at 37 ºC.

For cell uptake experiment, the harvested macrophages were employed. The macrophages were washed with sterile PBS to remove the non-adherent cells. After that NTZ dispersion and NTZ-TES at the concentration of 50 µg/mL were added into  96-well plate and incubated for 24 hr in controlled environment of 5% CO₂ and at 37 ºC. After 24 hr the supernatant was removed, and macrophages were washed twice with sterile PBS to remove the free drug. The attached macrophages were scraped off and centrifugation of cellular suspension was carried out at 4 ºC for 5 min to obtain pellets of macrophages. Methanol was added and sonicated for 5 min. The UV- spectroscopic examination was done to determine the NTZ content up taken by the macrophages.

Stability study

Stability study was conducted at temperatures 4 ± 2 ºC and 30 ± 2 ºC for the period of 4 months to evaluate the effects of different storage conditions on the optimized formulation. Samples stored at respective temperatures were evaluated for PS, PDI, ZP and the physical stability.