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Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 8 Number 12 (2019) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2019.812.072 An Experimental Study on the Effect of Pyrimethamine-Loaded Niosomes in the Treatment of Acute Toxoplasmosis Basma M. El-Mansory1*, Samy I. El-Kowrany1, Sirria M. El-Marhoumy1, Kholoud A. El-Nouby1, Mona A. Abd Elazeem2 and Gamal M. El Maghraby3 1 Department of Medical Parasitology, Faculty of Medicine, Tanta University, Tanta, Egypt 2 Department of Pathology, Faculty of Medicine, Tanta University, Tanta, Egypt 3 Department of Pharmaceutical Technology, Faculty of Pharmacy, Tanta University, Tanta, Egypt *Corresponding author ABSTRACT Keywords Toxoplasma gondii. Pyrimethamine. Niosomes. Nanostructures Article Info Accepted: 07 November 2019 Available Online: 10 December 2019 Toxoplasma gondii infection has a worldwide distribution. Pyrimethamine (PYR) is the most effective drug for treatment of toxoplasmosis. Unfortunately, it has low oral bioavailability which requires an increase in the dose that results in increased its side effects. Nanostructures showed promising potential to overcome this problem. This study aimed to evaluate the effect of intraperitoneal injection (IP) of PYR-loaded niosomes compared to PYR in the treatment of acute toxoplasmosis in experimentally infected mice. The study employed 240 mice that were divided into groups. Group I included Ia (20 uninfected untreated), Ib (20 infected untreated), Ic (non-infected and injected with placebo niosomes) and Id (20 non-infected mice injected with dimethyl sulfoxide). Groups II and III (a & b/each, 40 mice/each) were treated with PYR and PYR-loaded niosomes respectively in doses of 5 or 10 mg/kg/day for four successive days. Then all mice were sacrificed and their peritoneal fluids were examined by the scanning electron microscopy. Livers, spleens and brains were used for parasite count and for histopathological examination. This study showed that the niosomes improved the efficacy of PYR in the treatment of acute toxoplasmosis in mice. It was evidenced by increased the survival rate, decreased tachyzoites count, morphological changes of the tachyzoites and decreased inflammation. Niosomal PYR was effective at low dose with its efficacy being even greater than that of the solution even at high dose. It was concluded that PYR-niosomes formulation is a powerful alternative for reduction of PYR dose and its side effects. Introduction Toxoplasma gondii (T. gondii) infection has a worldwide distribution. Epidemiological surveys have suggested that up to one-third of the world’s population are infected with this parasite (Munoz et al., 2011). Man acquires the infection through ingestion of tissue cysts in undercooked or raw meat or by accidental ingestion of mature oocysts in the 542 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 contaminated food and drink. Congenital transmission is also another important route of infection that may lead to serious consequences. The infective stages of T. gondii include tachyzoite which is a rapidly dividing invasive stage, bradyzoite that is a slowly dividing in tissue cysts, and the sporozoite which is the environmental stage inside the oocyst. Tachyzoites are the dissemination forms that are able to invade nearly every nucleated cell in human body where they multiply in a parasitophorous vacuole (Robert-Gangneux and Dardé, 2012). These infective stages can invade the mucosa of the small intestine and multiply in the lymphoid macrophages in the lamina propria. They travel through lymphatics to invade the mesenteric lymph nodes where they proliferate before spreading by blood to all organs. In the immunocompetent hosts, the tachyzoites convert to chronic status forming bradyzoites which are encysted within the brain, eye and muscles. These cysts may remain dormant for life. When the immunity of the host decreases, the cysts rupture and the bradyzoites convert to the tachyzoites that invade new cells causing relapse (Hakeen 2010). This relapse can cause a life threatening systemic, ocular and most commonly neurological complications in these patients (Mordue et al., 2001; Pappas et al., 2009). Mortality due to toxoplasmic encephalitis (TE) in immunocompromised patients can reach 100% if treatment is delayed (Montoya et al., 2015). Pyrimethamine is highly effective in treatment of TE especially when used in combination with sulfadiazine. However, the combination of PYR with sulfadiazine can cause bone marrow suppression, hematological toxicity and life-threatening skin allergic reactions. These side effects are mainly due to sulfonamides (Dhapte et al., 2013). Thus, administration of PYR as a single drug may help to prevent these sulfonamide-associated side effects. It is highly selective for the tachyzoite form of Toxoplasma gondii with efficient brain parenchyma penetration (Katlama et al., 1996; Leport et al., 1992). Unfortunately, PYR suffers from poor and variable bioavailability after oral administration. Also, it is subjected to extensive hepatic metabolism which limits the amount of drug reaching the systemic circulation. Only 10% to 25% of serum concentrations of the drug can reach the cerebrospinal fluid (Montoya et al., 2015). This requires an increase in the dose to achieve the therapeutic blood and CSF levels which results in increased its side effects. Moreover, PYR is subjected to development of resistance which is aggravated by increasing the dose. So the use of nanostructures may improve the bioavailability of PYR that can result in clinical benefits (Patravale et al., 2004; Rabinow, 2004). Nanoparticles are colloidal submicron particles ranging from 1 nm to 100 nm in size (Biswas et al., 2014). Nanocarriers are potential drug delivery vehicles that can take the ultradisperse drugs to the specific intracellular targets, which are surrounded by complex physiological barriers (Pohlmann et al., 2013). The problems of solid nanostructures include the difficulty of preparation and poor physical stability which leads to particle size enlargement (Naseri et al., 2015). Niosomes are vesicular nanostructures that can encapsulate drugs irrespective to their physicochemical properties. They can be fabricated easily from non-ionic surfactants and cholesterol as the main components with minimal phospholipid being included in some cases. The properties can be further modified by manipulating the composition of niosomes. Peceol, a membrane fluidizing material has been added to niosomes to improve the oral bioavailability with the recorded positive results being explained on the base of 543 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 enhanced membrane permeability and/or preferential lymphatic transport (Sultan et al., 2016, 2018; Zoghroban et al., 2019). These vesicles have been shown to enhance the bioavailability of many drugs with promising data being recorded against parasites like Shistosoma in vitro and in vivo (Zoghroban et al., 2019). Preparation of the tested formulations Accordingly, the objective of this work was to investigate the effect of niosomal encapsulation of pyrimethamine on its efficacy against acute T. gondii infection in mice. Niosomes were prepared according to the composition presented in Table 1 (Sultan et al., 2016). Span 60, cholesterol, peceol and pyrimethamine were mixed before addition of ethanol. The mixture was heated on water bath to form clear dispersion. Water (0.75 ml) was added with mixing in the water bath until clarity. The clear liquid was removed from the water bath and cooled while mixing to form proniosomal gel. The remaining amount of water was added with continuous mixing to produce niosomes which were left to swell and hydrate for an overnight. Niosomes were then subjected to bath sonication for 30 minutes. Materials and Methods Parasite The RH virulent strain of Toxoplasma gondii was obtained from the Medical Parasitology Department, Faculty of Medicine, Alexandria University, Egypt. This was maintained at Medical Parasitology Department, Faculty of Medicine, Tanta University, Egypt by serial passages (at 3–4 days intervals) of tachyzoites intraperitoneally (IP) in Swiss albino mice (35 weeks old, 20 g weight). Pyrimethamine solution (1mg/ml) was prepared by dissolving the drug in dimethyl sulfoxide (DMSO) to produce 2 mg/ml. This solution was diluted with equal volume of distilled water to prepare the required concentration. Experimental animals The tachyzoites were collected from the peritoneal exudates of the mice on the 4th day of infection, washed three times and diluted with phosphate buffer saline (PBS), pH 7.4. These were used for infecting the mice at a dose of 3.5 X 103 tachyzoites/mouse according to Saudi et al., (2008). Two hundred and forty laboratory-bred male Swiss albino mice, aged 3–5 weeks and weighing 20–25 g were used. The mice were housed according to the guidelines of care and usage of animals for scientific purposes. Mice stool was examined microscopically to exclude the presence of parasites. This study was approved by the Ethics Committee of Tanta University, Egypt. Drugs Experimental groups Pyrimethamine powder (PYR) and Span 60 were obtained from Sigma-Aldrich, St. Louis, MO, USA. Peceol was obtained from Gattefosse, Saint- Priest Cedex, France. Cholesterol and ethanol were purchased from El-Nasr Pharmaceutical Chemicals Company, Cairo, Egypt. The mice were divided into three main groups Group I (Control group) This group included 80 mice that were subdivided equally into four subgroups: 544 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 Subgroup Ia Group III Subgroup Ia included 20 non-infected nontreated mice which were served as a control healthy group that were sacrificed at the beginning of the experiment for the comparative study. This group included 80 experimentally infected mice that were subdivided equally into two subgroups. These subgroups were treated with pyrimethamine-loaded niosmes (PYR-niosomes) in different doses according to Pissinate et al., (2014) as follow: Subgroup Ib Subgroup IIIa Subgroup Ib included 20 infected non-treated mice. Subgroup Ic Subgroup Ic included 20 non-infected mice that were injected intraperitoneally (IP) with 0.1 ml of non-medicated niosomes for four successive days. Subgroup IIIa included 40 mice which were injected IP with PYR-niosomes in a dose of 5 mg/kg/day. Subgroup IIIb Subgroup IIIb included 40 mice which were injected IP with PYR-niosomes in a dose of 10 mg/kg/day. Subgroup Id Subgroup Id included 20 non-infected mice that were injected intraperitoneally (IP) with 0.1 ml of DMSO for four successive days. Group II This group included 80 experimentally infected mice that were subdivided equally into two subgroups. These subgroups were treated with pyrimethamine (PYR) alone in different doses according to Pissinate et al., (2014) as follow: Subgroup IIa Subgroup IIa included 40 mice which were injected IP with PYR alone in a dose of 5 mg/kg/day. Subgroup IIb Subgroup IIb included 40 mice which were injected IP with PYR alone in a dose of 10 mg/kg/day. The treatment was initiated 24 hours postinfection in the studied groups and continued for four successive days according to Saudi et al., (2008). On the 5th day post infection, all the mice of the studied groups were anesthetized, sacrificed and subjected to the followings: Parasitological evaluation Survival rate estimation The survival rate was calculated in each group according to the following equation (Eissa et al., 2012) × 100 Parasite load The mean parasitic count was estimated in the Giemsa stained impression smears made from the livers, spleens and brains of the mice using the oil immersion objectives (×100) lens. The mean number of tachyzoites of ten different 545 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 fields from each studied organ of each mouse was calculated then the mean number of each infected subgroup was determined (Thiptara et al., 2006). Morphological study The peritoneal fluids of the studied groups were examined by the scanning electron microscopy (SEM) for detection of the morphological changes of T. gondii tachyzoites collected on the 5 th day post infection from each group. The peritoneal fluids were washed twice with PBS, and then fixed in glutaraldehyde. The specimens were washed 3 times by flooding with large volumes of sterile distilled water then processed according to Klainer and Betsch (1970), and examined using a Jeol-JSM-25 SII scanning microscope. Histopathological study Specimens from the livers, spleens and brains of the mice were processed for histological examination and examined to assess the presence of inflammation and the parasite. Statistical data analysis These data were analyzed by one-way ANOVA followed by Turkey’s multiple comparison as a post hoc test to determine significance of differences between groups using Statistical Package for Social Sciences (SPSS) (SPSS Inc., Chicago, Illinois, USA), software for windows, version (20). The difference was considered statistically significant when P < 0.05 according to Leslie et al., (1991). Results and Discussion Survival rate estimation The infected untreated group showed the lowest survival rate. Only 60% of the infected mice surviving on the 5th day post infection. Treatment with unprocessed pyrimethamine at doses of 5 and 10mg/kg/day increased the survival rate to 85%, 87.5% respectively. Treatment with noisome encapsulated drug at the same doses increased the survival rate to reach 90% and 95% respectively. Parasite load Biochemical study Urea and liver enzymes; aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured in the sera of the non infected control group Ic (injected IP with the non-medicated niosomes alone) and group Id (injected IP with DMSO) as compared to the healthy control group at the sacrifice time according to Sharma et al., (2003) and Paul et al., (2010). Figures 1-3 show representative light micrographs of the Giemsa-stained impression smears of liver, spleen and brain. The figures show the parasite load in these tissues before and after treatment with pyrimethamine or PYR-niosomes. The estimated numbers of the tachyzoites are presented in Table 2. There was a statistically significant reduction in the mean number of the Toxoplasma tachyzoites in the treated subgroups as compared to the infected control group in all studied organs. Ethics statement The study protocol was approved and conducted according to the guidelines of the Laboratory Animal Centre for Research Ethics Committee at Faculty of Medicine, Tanta University (code number: 31303/01/17). Also significant decrease (p<0.05) was detected between the subgroups IIa and IIb and the subgroups IIIa and IIIb. Also the difference between IIIa and IIb was significant in all studied organs (Table 2 and Fig. 1, 2 and 3). 546 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 Morphological study Figure 4 shows scanning electron micrographs of the morphological changes of tachyzoites recovered from the peritoneal exudates before and after treatment with pyrimethamine solution or niosomes. The tachyzoites recovered from the untreated group were of typical crescent shape with smooth regular surface. Treatment with PYR solution resulted in deformation of the tachyzoites which were distended and showed irregular ridges, deep furrows and disorganized conoid. This effect increased by increasing the dose. Treatment with PYR-niosomes resulted in higher degree of deformation with the recovered tachyzoites showed further increase in the severity of the deformation (Fig. 4). Histopathological study Figures 5-7 shows haematoxylin and eosin stained sections of livers, spleens and brains tissues recovered from uninfected mice, infected mice and infected treated mice. The uninfected mice showed normal histological features of each organ. The infected untreated group was characterized by marked inflammatory changes in the liver showing marked infiltration of portal tracts by inflammatory cells in addition to hydropic degeneration and fatty changes of hepatocytes. This was associated with congestion of central veins. Pseudocysts were also seen in the liver tissue reflecting the existence of large number of tachyzoites. Treatment with PYR solution reduced the inflammatory changes with the liver tissue showing moderate infiltration of portal tracts by inflammatory cells and congestion of central veins. Increasing the dose to10 mg/kg improved the picture which showed more reduction in these inflammatory changes. Treatment with PYR-niosomes resulted in further improvement of the histopathological features which showed mild infiltration of portal tracts by lymphocytes and mild congestion of the central vein. The efficacy was further fortified on using higher dose of PYR niosomes (Fig. 5). Regarding spleen sections, the infected control group showed hyperplastic lymphoid follicles, dilated congested sinusoids and marked infiltration by mono and multinucleated giant cells. Also Toxoplasma pseudocysts could be seen in the tissue. Treatment with PYR solution reduced the lymphoid follicles hyperplasia and congestion of the sinusoids while with increasing the dose of PYR there was a reduction in these changes in addition to decrease of the giant cells. Administration of PYR-niosomes resulted in a reduction in the lymphoid follicles hyperplasia. The dilatation of the congested sinusoids and their infiltration with a few giant cells became less evident. The higher dose of PYR-niosmes resulted in further improvement in the degree of inflammation (Fig. 6). Concerning brain sections, heavy inflammatory infiltrates and increased cellularity were observed in the infected untreated mice. The treatment of PYR decreased these inflammatory infiltrations in dose dependant manner while the use of PYRniosomes showed a mild degree of inflammation and the higher dose resulted in further reduction of the inflammatory infiltration (Fig. 7). Biochemical study Table 3 presents the biochemical levels of normal mice and those receiving placebo niosomes or DMSO. The results reveal no significant increase in the serum levels of urea and the liver enzymes (AST and ALT) in the after intraperitoneal administration of drug free niosomes or DMSO (Table 3). The treatment of toxoplasmosis is considered a huge challenge as there is no effective and 547 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 safe treatment up till now. The standard treatment is the combination of pyrimethamine (PYR) and sulfadiazine but this combined treatment is often associated with severe side effects which are intolerable by the patients (Anderson, 2005). This requires searching for and development of new drug candidates, a process which is very expensive and time consuming. Improvement of an existing drug is an alternative strategy with investigators concentrating on enhancing the bioavailability and tissue distribution of the target drugs. Colloidal nanostructures showed promising potential in this respect (Prieto et al., 2006). Niosomes are novel nanoscale drug carriers which have a high biological and physical stability that enable the delivery of the drug to its target site in a controlled manner (Mehta and Jindal 2014). Modulation of niosomal composition can tailor their characteristics with incorporation of peceol showing enhanced membrane permeability. Niosomes can travel through the lymphatic system before reaching the systemic circulation (Sultan et al., 2017). Accordingly, peceol containing niosomes were employed as nanocarrier for enhanced delivery of PYR against acute toxoplasmosis in mice. The efficacy of treatment was assessed by monitoring the survival rate, the parasite load in the liver, spleen and brain. In addition, morphological changes in the tachyzoites, histopathological changes and immunological response were also investigated. With respect to the survival rate, the infected untreated group showed the lowest survival rate. This rapid death of mice could be explained by the rapid and aggressive dissemination of the Toxoplasma tachyzoites in all cells of the mice (Sibley et al., 2002). The survival rate was increased after treatment with PYR solution in a dose dependent manner. This increase can be attributed to the inhibitory effect of PYR on tachyzoite replication with incomplete cure being attributed to low bioavailability of the drug. The superiority of niosomal PYR over the drug solution can imply better bioavailability from niosomes. Similarly, Pissinate et al., (2014) found that the lipid-core nanocapsules (LNC) loaded with PYR significantly improved the survival rate of the mice as compared to PYR alone. These results agree with that recorded in previous studies that proved that the use of nanosystems improved the efficacy of the antitoxoplasmic drugs and the survival rate in the treatment of acute toxoplasmosis in mice as Sordet et al., (1998), Schöler et al., (2001), Dunay et al., (2004) and Shubar et al., (2009 and 2011) and Anand et al., (2015). Regarding the parasite load, it could detect the density of the infection in tissues (El-Temsahy et al., 2002). There was a rapid dissemination of the parasite to the liver, spleen and brain of the infected control subgroup. This finding was in agreement with the studies done by ElTemsahy et al., (2002), Sibley et al., (2002), Eissa et al., (2012) and El Temsahy et al., (2016). There was a highly statistically significant reduction in the mean count of Toxoplasma tachyzoites in the treated subgroups as compared to the infected control group. PYR-niosomes were more effective in reducing the parasite load in all studied organs in the low dose of 5 mg/kg/day than PYR alone in the higher dose. This result agrees with Anand et al., (2015) who found that the use of the encapsulated bovine lactoferrin protein nanocapsules led to reduction of the parasite load in the mice acutely infected with toxoplasmosis. 548 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 Table.1 The composition of PYR-niosomes Material Span 60 Cholesterol Peceol Ethanol Water to Pyrimethamine Amount 0.6 gram(g) 0.15 g 0.15 g 0.75 g 25 ml 0.025 g Table.2 The mean count of Toxoplasma tachyzoites in the organs of the studied subgroups Organ Liver Spleen Brain Subgroups Mean ± SD P1 P2 P3 P4 Mean ± SD P1 P2 P3 P4 Mean ± SD P1 P2 P3 P4 Ib (n=12) 7.9 ± 1.43 IIa (n=34) 7.4 ± 1.43 0.079 IIb (n=35) 6.1 ± 0.65 0.001* 0.001* 4.3 ± 0.76 3.2 ± 0.53 0.001* 2.2 ± 0.91 0.001* 0.001* 2 ± 0.43 1.3 ± 0.43 0.001* 1.0 ± 0.66 0.001* 0.007* IIIa (n=36) IIIb (n=38) 1.5 ± 0.60 1.0 ± 0.48 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.012* 0.8 ± 0.33 0.5 ± 0.26 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.026* 0.4 ± 0.22 0.2 ± 0.18 0.001* 0.001* 0.001* 0.001* 0.001* 0.001* 0.037* *Significant (p< 0.05) Ib: T. gondii infected untreated mice. II: T. gondii infected mice treated IP with PYR alone in doses of 5 mg/kg/day (IIa) or 10 mg/kg/day (IIb). III: T. gondii infected mice treated IP with PYR-niosomes in doses of 5 mg/kg/day (IIIa) or 10 mg/kg/day (IIIb). P values: P1: Comparison with Ib +ve control; P2: Comparison of IIa with IIb, IIIa and IIIb. P3: Comparison of IIb with IIIa and IIIb; P4: Comparison of IIIa with IIIb. Table.3 Urea and liver enzymes (AST and ALT) levels in the serum of the control subgroups Biochemical parameter Urea (mean ± SD) AST (mean ± SD) ALT (mean ± SD) Ia (n=20) 27.65 ± 1.35 75.70 ± 18.47 47.93 ± 14.02 Ic (n=20) 28.02 ± 1.31 76.39 ± 12.52 50.76 ± 10.24 Id (n=20) 28.47 ± 1.65 80.48 ± 10.49 51.79 ± 7.09 *Significant (p < 0.05) Ia: non-infected untreated mice. Ic: non-infected mice that were injected IP with the niosomes alone in a dose of 5mg/kg/day. Id: non-infected mice that were injected IP with DMSO in a dose of 5mg/kg/day. P values: P1: comparison of Ia with Ic; P2: comparison of Ia with Id. 549 P1 0.421 0.879 0.411 P2 0.078 0.292 0.264 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 Fig.1 Giemsa stained liver impression smears of the mice of the studied subgroups (x1000) a T. gondii infected untreated mice (Ib). b T. gondii infected mice treated with PYR 5 mg/kg/day (IIa). c T. gondii infected mice treated with PYR 10 mg/kg/day (IIb). d T. gondii infected mice treated with PYR-niosomes 5 mg/kg/day (IIIa). e T. gondii infected mice treated with PYRniosomes 10 mg/kg/day (IIIb) 550 Int.J.Curr.Microbiol.App.Sci (2019) 8(12): 542-561 Fig.2 Giemsa stained spleen impression smears of the mice of the studied subgroups (x1000) a T. gondii infected untreated mice (Ib). b T. gondii infected mice treated with PYR 5 mg/kg/day (IIa). c T. gondii infected mice treated with PYR 10 mg/kg/day (IIb). d T. gondii infected mice treated with PYR-niosomes 5 mg/kg/day (IIIa). e T. gondii infected mice treated with PYRniosomes 10 mg/kg/day (IIIb) 551