Materials
PZQ (Gift of the Egyptian International Pharmaceuticals Industries Company (EIPICO), Cairo, Egypt), MFS (Chem-Impex International, New York, USA), Labrafac® lipophile WL 1349 (Gattefossé SA, Saint-Priest, France), Kolliphor HS 15 (BASF, Ludwigshafen, Germany), Lipoid S100 (a soybean lecithin containing 94% of phosphatidylcholine, Lipoïd GMBH, Ludwigshafen, Germany), Oleic acid (OA, Sigma-Aldrich Co., St Louis, MO, USA), acetonitrile HPLC grade (Thermo Fisher Scientific, Waltham, MA, USA), Span® 80 (LobaChemie for Laboratory Reagents and Fine Chemicals, Mumbai, India). All other chemicals were of analytical grade.
Formulation and characterization of lipid nanocapsules
LNCs were formulated with oleic acid and Span 80 and prepared by the phase inversion method (24, 27). In brief, Kolliphor® HS 15, Labrafac lipophile WL 1349 and deionized water containing NaCl (0.88% w/w of the final dispersion) were weighed and mixed using a magnetic stirrer in the ratio 5:6:9. Span 80 (2% w/w) and oleic acid (6 % w/w) were added to the primary mixture which was subjected to three progressive heating and cooling cycles between 45-75°C at 4°C/min. An irreversible shock was induced by two-fold dilution with cold deionized water (0-2°C) added to the formed o/w emulsion at a temperature 1-3°C from the beginning of the phase inversion zone. This was followed by slow magnetic stirring at room temperature for five min. For the preparation of drug loaded LNCs, PZQ was added to the primary mixture of ingredients at concentrations 25 or 12.5 mg/ml, whereas MFS was added just before quenching at a concentration of 2 or 1 mg/ml of the final dispersion. The procedure was used to prepare a higher fixed dose combination containing 25 mg PZQ and 2 mg MFS/ml of dispersion and a lower fixed dose combination containing 12.5 mg PZQ and 1 mg MFS/ml of dispersion and their corresponding singly loaded counterparts, PZQ 25 mg/ml, MFS 2 mg/ml, PZQ 12.5 mg/ml and MFS 1 mg/ml. The fixed dose combination LNC dispersions were used in calculated volumes to provide a dose of 250 mg/kg PZQ-20 mg/kg MFS and 125 mg/kg PZQ-10 mg/kg MFS respectively in the antischistosomal study in mice.
LNC formulations were characterized for morphology, colloidal properties and drug entrapment efficiency (EE%). The morphology of LNCs was examined by transmission electron microscopy (TEM) using JEOL, JEM-100 CX Electron Microscope, Tokyo, Japan. Before analysis, the LNC dispersion was treated with 2% w/v uranyl acetate solution as a negative stain and sprayed onto copper grids. Shots were taken at X 7500 at 80 kV. The average particle size, polydispersity index (PdI) and zeta potential (ZP) were measured by photon correlation spectroscopy (PCS) at a fixed angle 173° using a 4 mW He-Ne laser at 25ºC. The EE% was obtained by determining the concentration of free (unentrapped) PZQ and MFS in the ultrafiltrate after separation of LNCs using an ultrafiltration/centrifugation technique. The concentration of unentrapped PZQ in the ultrafiltrate was determined by HPLC-UV as reported (24). MFS concentration was measured by a modified spectrophotometric assay originally reported for quaternary ammonium compounds and validated for MFS quantitation (16).
Antischistosomal efficacy in mice
Schistosoma mansoni infection of animals
The life cycle of S. mansoni was maintained in the Medical Parasitology Department, Faculty of Medicine, Alexandria University by serial passages in laboratory-bred Biomphalaria alexandrina snails and Swiss strain albino mice as reported . A total of 128 mice, six to eight weeks old, weighing 20-30 gram each, were obtained from the animal house of the Medical Parasitology Department, Faculty of Medicine, Alexandria University. Mice were housed under specific pathogen-free barrier conditions. Each mouse was infected with 100 ±10 freshly shed cercariae using the paddling technique (40). All mice were subjected to infection.
Animal groups
One hundred and twenty-eight mice were allocated to a nontreated control group (NT) including 8 mice and 2 experimental groups, Group I and Group II, 60 mice each.
Group I: subdivided into 3 subgroups 20 mice each and treated with LNC dispersions corresponding to the required dose of the higher fixed dose nanocombination or its singly loaded counterpart controls as follows:
Subgroup Ia: PZQ 250 mg/kg
Subgroups Ib: MFS 20 mg/kg
Subgroup Iab: PZQ 250 mg-MFS 20 mg /kg
Group II: subdivided into 3 subgroups 20 mice each and treated with LNC dispersions corresponding to the required dose of the lower fixed dose nanocombination or its singly loaded counterpart controls as follows:
Subgroup IIa: PZQ 125 mg/kg
Subgroup IIb: MFS 10 mg/kg
Subgroup IIab: PZQ 125-MFS 10mg /kg.
Mice in all treated subgroups (Ia, Ib and Iab, IIa, IIb and IIab) were administered a calculated volume of the LNCs dispersion corresponding to the required dose of the PZQ-MFS fixed dose combinations or the corresponding singly loaded LNCs by gastric gavage. Mice in these groups were further subdivided into three subgroups (1, 2, 3) which were given a single oral dose of the drug(s) nanoformulations at three different dates (on the initial day of infection, 21st and 42nd days p.i.) corresponding to the three stages of S. mansoni life cycle (invasive, juvenile and adult stages (41). The number of mice/ group treated on the initial day of infection and 21st day p.i. (against invasive and immature stages) was six whereas, it was eight for all other subgroups treated on the 42nd day p.i. (against the adult stage). Two infected mice treated on the 42nd day p.i. were sacrificed 24 h after administration of the nanoformulations to collect the adult worms for morphological examination using SEM (Jeol JSM- IT200, Jeol, Tokyo, Japan). The remaining mice of all subgroups were sacrificed on the 49th day p.i. The therapeutic efficacy of the two fixed dose nanocombinations in comparison with the corresponding singly loaded control LNCs and the nontreated control was assessed by determination of the percentage reduction in total worm burden, the size of hepatic granulomas, histopathological changes in liver parenchyma and examination of the morphology of recovered worms by SEM.
Antischistosomal activity assessment
Morphological examination by scanning electron microscopy
Adult worms were recovered 24 h post administration of nanoformulations from two infected mice from all subgroups treated on the 42nd day p.i. against the adult stage for SEM imaging. Worms were fixed in cold 2.5% buffered glutaraldehyde phosphate (pH 7.4), processed, examined under SEM and photographed (42).
Estimation of adult worm burden
On the 49th day p.i., adult worms were recovered from the hepatic and mesenteric vessels from mice in all study groups using the perfusion technique (40).
Histopathological examination
Specimens of the liver of mice of all study groups were fixed in 10% neutral buffered formalin. Histological sections, 5 μm-thick, were stained with hematoxylin and eosin (H&E). Pathological changes in the hepatic parenchyma were observed and the mean size of granulomas was determined. Only granulomas containing one central clearly identifiable egg were selected (43).
Statistical analysis
Data were analyzed using IBM SPSS software package version 20.0 (Armonk, NY: IBM Corp). Kolmogorov-Smirnov test was used to verify the normality of distribution. Quantitative data were described using mean and standard deviation. Significance of the obtained results was judged at the 5% level. For normally distributed quantitative variables, F-test (ANOVA) was used to compare between more than two groups and Post Hoc test (Tukey) was used for pairwise comparisons. The percentage reduction (% R) of the adult worm load as well as the granuloma size were calculated as follows:
Where, (N) is the mean number of worms or the mean granuloma size in the infected nontreated group and (n) is the mean number of worms or the mean granuloma size in the infected treated subgroups.