DOI: 10.5061/dryad.cfxpnvx7d
title: Macrophage targeting of nitazoxanide-loaded transethosomal gel in cutaneous leishmaniasis

description: 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.
description: <strong>Preparation of
 transethosomes</strong> 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.
 <strong>Preparation of nitazoxanide loaded transethosomal
 gel (NTZ-TEG)</strong> 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. <strong>NTZ-TES PS, PDI, ZP
 determination and TEM analysis</strong> 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
 <strong>In vitro release and kinetic drug release study
 </strong> 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 <strong>Ex vivo permeation and skin
 deposition study</strong> 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 <strong>In vitro leishmanicidal
 activity against promastigotes</strong> 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
 1×10<sup>6</sup> promastigotes/mL in each well of 96-well
 plate having 20 µL samples, at the concentration of 100 µg/mL (having
 &lt;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 IC<sub>50</sub> was
 calculated by using GraphPad prism software.
  The percentage growth inhibition was then
 calculated by following equation <sup>% Growth Inhibition = Optical
 density of control - optical density of test<strong> /
 </strong>optical density of control X
 100</sup>
 <strong>Ex vivo cell uptake study</strong>
 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 × 10<sup>4
 </sup>million cells/well and incubated for 24 hours with 5 %
 CO<sub>2</sub> 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% CO<sub>2</sub> 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. <strong>Stability
 study</strong> 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.