Histopathological and biochemical assessment of the therapeutic effect of gold nanoparticles on experimental chronic toxoplasmosis

Abstract

Background: Toxoplasmosis is a zoonotic disease caused by the intracellular opportunistic protozoan, T. gondii that infects both vertebrate and invertebrate cells. Nanoparticles (NPs) provide promising therapeutic agents for effective treatment of parasitic diseases, by overcoming the drawbacks of low bioavailability and poor cellular permeability of anti-parasitic drugs. Objective: To assess the therapeutic effect of gold (Au) NPs on experimentally infected mice with chronic toxoplasmosis. Material and Methods: Sixty-five laboratory-bred female Swiss albino mice were included. Five mice were left as control negative (non-infected non-treated), while the rest were experimentally infected orally with avirulent T. gondii strain (ME49). Fifty days post infection (pi), infected mice were divided into 4 groups (15 mice each); group 1: control group (infected non-treated); group 2: treated with Spiramycin (Rovamycin); group 3: treated with low-dose AuNPs; and group 4: treated with high-dose AuNPs. Treatment was administered orally for 10 days. All mice were sacrificed sixty days pi. Assessment of the therapeutic effect of AuNPs was achieved using parasitological, histopathological and biochemical parameters. The first included estimation of the parasite tissue cysts numbers and size in impression smears from brain, liver and spleen; histopathological parameters evaluated inflammation, necrosis and hemorrhages in tissues. Liver transaminases: aspartate transaminase (AST) and alanine transaminase (ALT) were measured in sera of all groups as a biochemical parameter. Results: Statistically significant reductions were observed in the mean cyst count and size in brain, liver, and spleen of the infected treated group with high dose AuNPs (group 4) compared to the other infected groups. Histopathological examination of the brain, liver and spleen tissues showed that inflammatory reaction was decreased in both AuNPs-treated groups in comparison to Spiramycin-treated group and the non-treated mice. There was a significant reduction in the mean values of ALT and AST in mice treated with AuNPs high dose in comparison to other groups (P<0.05). Conclusion: AuNPs have a potentially therapeutic effect especially the high dose against experimental chronic toxoplasmosis. PARASITOLOGISTS UNITED JOURNAL 172 Traditional chemicals or natural compounds that have limited access to the CNS and are of high toxicity to the host, are used in current therapeutic regimens. To treat these infections and allow the medication to cross the blood-brain barrier, improvements in drug administration and formulation are needed[9]. Toxoplasmosis is treated with a mixture of pyrimethamine and sulfadiazine that despite their sufficient efficacy, can have common toxic side effects such as allergy, bone marrow suppression, folic acid deficiency, and hematologic toxicity[10]. Spiramycin is a bacteriostatic macrolide that has long been demonstrated to be effective against toxoplasmosis in mice[11]. Anti-toxoplasmic activity of Spiramycin was tested in murine infection models using T. gondii virulent (RH) and avirulent (ME 49) strains. Treatment with a dose of 100 or 200 mg/kg/day showed minor side effects[12]. Several factors, including membrane permeability and solubility, influence the bioavailability of any drug, including Spiramycin[13]. Studies were promoted to improve the solubility and dissolution rate of drugs with low water solubility[14]. Nanotechnology is the study of small materials with diameters ranging from a few nanometers to hundreds of nanometers. By affecting many biomolecules on the cell surfaces and intracellularly, this size can alter several physicochemical and biochemical properties of the cells. Accordingly, the use of nanotechnology in the treatment, tracking, prevention, and control of biological diseases is known as nanomedicine[15]. Nanomedicine applications use a variety of nanomaterials, including metals, lipids, and polymers, which are made up of various materials[16]. Because of their small size, NPs can pass through membrane barriers, resulting in increased reactivity[17]. Metal NPs with anti-parasitic properties, such as silver and gold, are of particular interest for this reason[18]. Therefore, this study aims to assess the therapeutic effect of AuNPs on experimentally infected mice with chronic toxoplasmosis. MATERIAL AND METHODS This experimental study was conducted from September to November 2019, at the National Research Centre, Cairo, Egypt. Study design: The study included five groups; two control groups (non-infected non-treated, and infected non-treated), and three infected treated groups. Fifty days pi, mice were orally treated with Spiramycin, lowdose and high-dose AuNPs for 10 days. Sixty days pi, all mice were sacrificed and the therapeutic effect of AuNPs was assessed using parasitological, histopathological, and biochemical parameters. Animals and T. gondii strain: Sixty-five laboratorybred female Swiss Albino mice, 6-8 weeks old; ~20-25 g in weight, were purchased from the animal house, National Research Centre, Cairo, Egypt. Mice were housed in well ventilated cages and were fed standard pellet food with free access to water. The ME49 strain was obtained from the batch maintained at the Zoonotic Diseases Department, National Research Center, Cairo, Egypt. Sixty mice were experimentally infected orally with 20 cysts/mouse. Five mice served as control negative group and their serum samples were used for determination of the liver enzymes levels. Gold NPs preparation: AuNPs was purchased from NanoTech Egypt company for Photo-Electonics, Cairo, Egypt and was prepared by chemical reduction method[19]. Briefly, HAuCl4 solution was used as Au3+ ions precursor; while sodium citrate was used as mild reducing and stabilizing agent. Using transmission electron microscopy, properties of AuNPs were determined as sperical, water soluble particles of ~ 15±5 nm, with average size (λ max = 520±3 nm), and 196 ppm concentration in its liquid form (Figure 1). Fig. 1. AuNPs properties by transmission electron microscopy (TEM). Animal grouping: Fifty days pi, infected mice were randomly divided into 4 equal groups (15 mice each). Group 1 included non-treated control positive mice. Groups 2, 3 and 4 were treated orally by esophageal tube for 10 days with Spiramycin (100 mg/kg/day), low dose (400 μg/kg/day) and high dose (800 μg/kg/day) of AuNPs, respectively. Impression smears: Estimation of T. gondii cysts count and size in impression smears[20] stained with Giemsa was performed from brain, liver and spleen of all infected mice. Three smears of each organ/mouse were examined using oil immersion and the mean of 10 different fields was calculated[21-22]. Histopathological assessment: Sections from brain, liver and spleen of G 1-4 infected with T. gondii were examined after staining with Haematoxylin and Eosin (HE)[23]. Biochemical assessment: After sacrifice of all mice including negative control group, one ml blood sample was obtained from each mouse to measure liver transaminases; AST and ALT levels. Enzymes were colorimetrically determined in sera of the all groups using spectrophotometery (Janway, Keison Products, UK)[24]. The kits were obtained from Spectrum, Cairo, Egypt). Nanomediciene in treatment of chronic toxoplasmosis Ahmed et al., 173 Table 1. Comparisons between cysts count in brain, liver, and spleen of the study groups. Organ Groups Mean count ± SD (SE) Statistical analysis 95% confidence interval@ P value# Brain Control Spiramycin AuNPs (low) AuNPs (high) Total 18.93 ± 1.10 (0.63) 8.20 ± 0.52 (0.30) 3.00 ± 0.34 (0.20) 0.13 ± 0.11 (0.06) 7.56 ± 7.50 (2.16) 16.19 21.67 6.88 9.51 2.13 3.86 0.15 0.42 2.79 12.34 0.000 Liver Control Spiramycin AuNPs (low) AuNPs (high) Total 20.46 ± 1.70 (0.98) 7.86 ± 0.30 (0.17) 3.20 ± 0.20 (0.11) 0.26 ± 0.11(0.06) 7.95 ± 8.09 (2.33) 16.24 24.69 7.10 8.62 2.70 3.69 0.02 0.55 2.80 13.1 0.000 Spleen Control Spiramycin AuNPs (low) AuNPs (high) Total 19.80 ± 1.40 (0.8) 8.06 ± 0.60 (0.8) 3.33 ± 0.11 (0.06) 0.20 ± 0.20 (0.11) 7.85 ± 7.80 (2.25) 16.32 23.27 6.46 9.66 3.04 3.62 0.29 0.69 2.89 12.80 0.000 Statistical analysis: Data were organized, tabulated, and statistically analyzed using SPSS version, 16.00. For quantitative data, mean, percentage and standard deviation were calculated. ANOVA test was used to compare variables between groups. Results were considered significant when P value was < 0.05. Ethical statement: The present study was approved from Research and Ethic committee of the Faculty of Medicine, Sohag University, Egypt. Animal experiments complied with the Egyptian national regulations according to the procedures for using laboratory animals and in accordance with the internationally accepted principles for laboratory animal use and care.