The Anti-schistosomal Activity of Magnetite and Zero-valent Iron Nanoparticles on Schisosoma mansoni: AN in Vivo Study

Background: Depending mainly on Praziquantel (PZQ) for treatment of schistosomiasis poses a great challenge in terms of effectiveness and resistance. Nanoscale particles formed by metals as iron nanoparticles (INPs) have recently gained approval from the Food and Drug Administration for use as therapeutic agents. Therefore, INPs application as potential therapeutic agents against schistosomiasis may give promising results. Objective: The present study aimed at assessing the efficacy of INPs; iron oxide or magnetite INPs (MNPs) and zero-valent INPs (ZV-INPs) on S. mansoni using parasitological and histopathological parameters. Material and Methods: In the current study, MNPs and ZV-INPs were prepared by biogenic synthesis and were given to mice orally on the 42nd day post infection (dpi) with S. mansoni in a dose of 10 mg/kg for four consecutive doses. The therapeutic efficacy was assessed using parasitological (mortality rate, adult worm load as well as female fecundity) and histopathological parameters (tissue egg count in both liver and intestine) in comparison to untreated and PZQ treated control groups. Results: Results revealed that ZV-INPs have a significant effect in decreasing both tissue egg count and hepatic granulomata size. While the MNPs have a significant effect against the total and female worms burden, tissue egg counts, female fecundity, and number of liver granuloma. Conclusion: Herein, it was concluded that both types of INPs used in the study are potentially effective anti-schistosomal agents. PARASITOLOGISTS UNITED JOURNAL 270 viability in a dose-dependent manner. Third, the most important factor of NPs toxicity is their stability, both in vivo and during synthesis and storage[7-9]. Non-oxidized ZV-INPs are widely used in environmental research due to their ability to produce high-energy reactive oxygen species. The latter can overcome and degrade organic pollutants that are non-decomposable from the environment. In the field of medicine, the incorporation of ZV-INPs with silver targeting malignant cells, led to malignant cell apoptosis and autophagy[6]. On the other hand, magnetic NPs, especially iron oxide or MNPs, became of new interest for scientists due to the phenomenon of super-paramagnetism. They obey the Coulomb’s law of electrostatic force interaction[10], where they can be directed to active sites in vivo under the influence of an external electromagnetic field[11]. Hence, ferrimagnetic iron oxide NPs gain medical interest especially in the field of diagnostics. In the field of therapeutics, ferumoxytol is a newly modified product that is approved for treatment of anemia[12], and is considered as a promising drug in treatment of cancer due to its effect on macrophage polymerization[13]. In the field of infectious diseases, MNPs have shown promising potential for the delivery of certain bactericidal agents to highly restricted microenvironments[14]. This mainly depends on the phenomenon of magnetic fluid hyperthermia, i.e., the application of a fluctuating magnetic field makes magnetic NPs dissipate energy as heat, causing a localized increase in temperature around them[15]. However, several studies proved that the intrinsic antimicrobial properties of MNPs and ZV-INPs are due to the generation of reactive oxygen species that damage microbes’ DNA, RNA and proteins[16-18]. In the scope of medical parasitology, superparamagnetic iron oxide NPs introduced a new non-invasive tracking technique for E. histolytica trophozoites in vivo[19]. Besides, MNPs showed their effectiveness in separation of P. falciparum infected erythrocytes from non-infected ones[20]. Iron oxide in the form of beads showed acceptable sensitivity and feasibility in diagnosis of schistosomiasis through the detection of Schistosoma antigens using ELISA[21]. At the therapeutic level, magnetic fluid hyperthermia proved to play a role as an action mechanism of NPs against the protozoan parasite L. mexicana in vitro[22]. Finally, INPs were previously tested in vitro on adult S. mansoni worms and results showed several tegumental derangements revealed by scanning electron microscopy. Besides, the NPs proved to increase the mortality rate of B. alexandrina snails[23]. Thus, we designed the present study to interpret the efficacy of both ZV-INPs and MNPs in comparison to PZQ against experimental schistosomiasis mansoni, using parasitological and histopathological parameters. MATERIAL AND METHODS This case-control experimental study was conducted during the period from January to April 2021. Mice infection and all parasitological and histopathological assessments were performed at the laboratory of Medical Parasitology Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt. Biosynthesis of INPs was performed in the Laboratory of the City of Scientific Research and Technological Applications (SRTA-City), Alexandria. Study design: Forty mice were experimentally infected with S. mansoni cercaria, and, were equally divided into four groups (Table 1). Stool examination was performed to confirm absence of any other parasitic infection before starting the treatment. Iron NPs were prepared by biogenic synthesis due to its safety and low expense when compared to traditional synthesis methods. A pilot study was conducted to determine the least effective dose of MNPs and ZV-INPs capable of decreasing the total worm burden, and 10 mg/kg/d for four consecutive days was selected. Both PZQ and INPs were given orally starting from the 42nd dpi. All animals were sacrificed on the 49th dpi. Parameters used to evaluate INPs therapeutic efficacy in comparison to PZQ included parasitological and histopathological assessments. Parasite and snails: The Egyptian strain of S. mansoni was used in the current study. Twenty shedding adult B. alexandrina snails (4-6 mm in diameter) were obtained from the Schistosome Biological Supply Centre, Theodor Bilharz Research Institute, Cairo, Egypt. Snails were allowed to shed under light and the fresh exiting cercariae were used to infect the mice. Each mouse was infected with 100 freshly shed cercariae using the paddling technique[24]. Experimental animals: Forty male Swiss strain albino mice, four to six weeks old, weighing 20-25 grams each were obtained from the animal house of the Medical Parasitology Department, Faculty of Medicine, Alexandria University. Egypt. Mice were kept in a Groups Characteristics I II III IV Infected, and non-treated. Infected and treated orally with PZQ in a dose of 500 mg/kg once. Infected and treated with ZV-INPs (10 mg/kg/d) for four consecutive days. Infected and treated with MNPs (10 mg/kg/d) for four consecutive days. Table 1. Study groups of mice. MNPs: Magnetite INPs; PZQ: Praziquantel; ZV-INPs: Zero-valent INPs Effect of iron nanoparticles on Schistosoma Younis et al., 271 pathogen-free environment with standard conditions of light and temperature. They were fed on bread and milk in alternation with wheat. The animals had free access to food and water. Tested agents PZQ: Distocide TM (EIPICO, Egypt), 600 mg tablets, was purchased from the local pharmacy. One tablet was crushed before use and dissolved in 6 ml 60% ethyl alcohol to obtain a solution with a concentration of 100 mg/ml. On the 42nd dpi, each mouse in group II received 0.1 ml of the prepared solution containing 10 mg of PZQ orally by gavage using a ball-tipped feeding needle (i.e., total dose of 500 mg/kg)[25]. Biosynthesis of INPs: The biogenic synthesis of MNPs and ZV-INPs was performed under aerobic and anaerobic conditions by Proteus mirabilis strain 10B as previously described[26,27]. Characterization of INPs: To identify the criteria of the biologically synthesized MNPs and ZV-INPs, the following assessments were conducted[28,29]. First, measurement of absorption spectra was done by UVVis spectrophotometer (Labomed model UV-Vis double beam spectrophotometer, USA). Second, determination of the crystalline nature was identified by X-ray diffractometer (Shimadzu 7000, USA). Third, description of the size and morphology was accomplished by transmission electron microscope (JEOL JEM-1230, Japan). Fourth, study of the magnetic features was performed by vibration sample magnetometry (VSM). Finally, measurement of electrostatic potential with polydisperisty index (PDI) was recorded by Zetasizer Nano (Malvern Instruments, Worcestershire, UK). Administration of INPs: On the 42nd dpi, each mouse in groups III and IV received 0.2 ml of the prepared suspension composed of 1 mg/ml of ZV-INPs and MNPs, respectively. Mice were inoculated orally by gavage using a ball-tipped feeding needle for four consecutive days. Parasitological assessment: Mice infection was confirmed by stool examination starting from the 35th dpi. The parasitological assessment included the estimation of the total count of adult worms, female worm load, tissue egg count in both liver and intestine, as well as female fecundity. Adult worms were recovered from the hepatic and mesenteric vessels by perfusion technique[30] to assess adult worm burden after mice sacrifice on the 49th dpi[31]. All mice were injected with 500 units of heparin and then anaesthetized by IV injection of an overdose (150 ml/ kg) of thiopental sodium[32]. Adult worms recovered from the hepatic and portomesenteric vessels were counted[33]. To determine the tissue egg count, parts of the liver and intestine from each mouse were weighed, cut into small pieces, then artificially digested by 10 ml of 4% potassium hydroxide for each gram of tissue. The containers were covered and left overnight at room temperature to ensure complete tissue digestion without egg destruction. After thorough shaking, eggs present in 0.1 ml of the tissue suspension were counted five times on five separate slides. To determine the number of eggs/one ml of the digestive fluid, the sum of the five readings was multiplied by 20 to obtain the egg count in ten ml of fluid representing th